Antibacterial agents

ABSTRACT

Compounds of formula (I) are antibacterials:  
                 
 
     wherein:  
     R 1  represents hydrogen, or C 1 -C 6  alkyl or C 1 -C 6  alkyl substituted by one or more halogen atoms; R 2  represents a group R 10 —(X) n —(ALK) m — wherein R 10  represents hydrogen, or a C 1 -C 6  alkyl, C 2 -C 6  alkenyl, C 2 -C 6  alkynyl, cycloalkyl, aryl, or heterocyclyl group, any of which may be unsubstituted or substituted by (C 1 -C 6 )alkyl, (C 1 -C 6 )alkoxy, hydroxy, mercapto, (C 1 -C 6 )alkylthio, amino, halo (including fluoro, chloro, bromo and iodo), trifluoromethyl, cyano, nitro, —COOH, —CONH 2 , —COOR A , —NHCOR A , —CONH A , —NH A , —NR A R B , or —CONR A R B  wherein R A  and R B  are independently a (C 1 -C 6 )alkyl group, and ALK represents a straight or branched divalent C 1 -C 6  alkylene, C 2 -C 6  alkenylene, or C 2 -C 6  alkynylene radical, and may be interrupted by one or more non-adjacent —NH—, —O— or —S—linkages, X represents —NH—, —O— or —S—, and m and n are independently 0 or 1; and A represents a group as defined in the specification.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation application of pending U.S.application Ser. No. 09/355,489, filed Jan. 7, 2000, entitled“Antibacterial Agents” which is hereby incorporated by reference in itsentirety.

FIELD OF THE INVENTION

[0002] This invention relates to the use of N-formyl hydroxylaminederivatives as antibacterial agents, to a novel class of such compounds,and to pharmaceutical and veterinary compositions comprising suchcompounds.

BACKGROUND TO THE INVENTION

[0003] In General, bacterial pathogens are classified as eitherGram-positive or Gram-negative. Many antibacterial agents (includingantibiotics) are specific against one or other Gram-class of pathogens.Antibacterial agents effective against both Gram-positive andGram-negative pathogens are therefore generally regarded as having broadspectrum activity.

[0004] Many classes of antibacterial agents are known, including thepenicillins and cephalosporins, tetracyclines, sulfonamides,monobactams, fluoroquinolones and quinolones, aminoglycosides,glycopeptides, macrolides, polymyxins, lincosamides, trimethoprim andchloramphenicol. The fundamental mechanisms of action of theseantibacterial classes vary.

[0005] Bacterial resistance to many known antibacterials is a growingproblem. Accordingly there is a continuing need in the art foralternative antibacterial agents, especially those which have mechanismsof action fundamentally different from the known classes.

[0006] Amongst the Gram-positive pathogens, such as Staphylococci,Streptococci, Mycobacteria and Enterococci, resistant strains haveevolved/arisen which makes them particularly difficult to eradicate.Examples of such strains are methicillin resistant Staphylococcus aureus(MRSA), methicillin resistant coagulase negative Staphylococci (MRCNS),penicillin resistant Streptococcus pneumoniae and multiply resistantEnterococcus faecium.

[0007] Pathogenic bacteria are often resistant to the aminoglycoside,-lactam (penicillins and cephalosporins), and chloramphenicol types ofantibiotic. This resistance involves the enzymatic inactivation of theantibiotic by hydrolysis or by formation of inactive derivatives. Theβ-lactam (penicillin and cephalosporin) family of antibiotics arecharacterised by the presence of a β-lactam ring structure. Resistanceto this family of antibiotics in clinical isolates is most commonly dueto the production of a “penicillinase” (β-lactamase) enzyme by theresistant bacterium which hydrolyses the β-lactam ring thus eliminatingits antibacterial activity.

[0008] Recently there has been an emergence of vancomycin-resistantstrains of enterococci (Woodford N. 1998 Glycopeptide-resistantenterococci: a decade of experience. Journal of Medical Microbiology.47(10):849-62). Vancomycin-resistant enterococci are particularlyhazardous in that they are frequent causes of hospital based infectionsand are inherently resistant to most antibiotics. Vancomycin works bybinding to the terminal D-Ala-D-Ala residues of the cell wallpeptidioglycan precursor. The high-level resistance to vancomycin isknown as VanA and is conferred by a genes located on a transposableelement which alter the terminal residues to D-Ala-D-lac thus reducingthe affinity for vancomycin.

[0009] In view of the rapid emergence of multidrug-resistant bacteria,the development of antibacterial agents with novel modes of action thatare effective against the growing number of resistant bacteria,particularly the vancomycin resistant enterococci and β-lactamantibiotic-resistant bacteria, such as methicillin-resistantStaphylococcus aureus, is of utmost importance.

BRIEF DESCRIPTION OF THE INVENTION

[0010] This invention is based on the finding that certain N-formylhydroxylamine derivatives have antibacterial activity, and makesavailable a new class of antibacterial agents. The inventors have foundthat the compounds with which this invention is concerned areantibacterial with respect to a range of Gram-positive and Gram-negativeorganisms. Furthermore, there is evidence that some compounds areantibacterial with respect to bacteria which are resistant to commonlyused antibiotics such as vancomycin and the β-lactam antibiotics, forexample methicillin-resistant Staphylococcus aureus.

[0011] Although it may be of interest to establish the mechanism ofaction of the compounds with which the invention is concerned, it istheir ability to inhibit bacterial growth which makes them useful.However, it is presently believed that their antibacterial activity isdue, at least in part, to intracellular inhibition of bacterialpolypeptide deformylase (PDF) enzyme.

[0012] Bacterial polypeptide deformylases (PDF) (EC_(3.5.1.31)), are aconserved family of metalloenzymes (Reviewed: Meinnel T, Lazennec C,Villoing S, Blanquet S, 1997, Journal of Molecular Biology 267, 749-761)which are essential for bacterial viability, their function being toremove the formyl group from the N-terminal methionine residue ofribosome-synthesised proteins in eubacteria. Mazel et al. (EMBO J. 13(4):91 4-923, 1994) have recently cloned and characterised an E. coliPDF. As PDF is essential to the growth of bacteria and there is noeukaryotic counterpart to PDF, Mazel et al. (ibid), Rajagopalan et al.(J. Am. Chem. Soc. 119:12418-12419, 1997) and Becker et al., (J. BiolChem. 273(19):11413-11416, 1998) have each proposed that PDF is anexcellent anti-bacterial target.

[0013] Certain N-formyl hydroxylamine derivatives have previously beenclaimed in the patent publications listed below, although very fewexamples of such compounds have been specifically made and described:EP-B-0236872 (Roche) WO 92/09563 (Glycomed) WO 92/04735 (Syntex) WO95/19965 (Glycomed) WO 95/22966 (Sanofi Winthrop) WO 95/33709 (Roche) WO96/23791 (Syntex) WO 96/16027 (Syntex/Agouron) WO 97/03783 (BritishBiotech) WO 97/18207 (DuPont Merck) WO 98/38179 (GlaxoWellcome) WO98/47863 (Labs Jaques Logeais)

[0014] The pharmaceutical utility ascribed to the N-formyl hydroxylaminederivatives in those publications is the ability to inhibit matrixmetalloproteinases (MMPs) and in some cases release of tumour necrosisfactor (TNF), and hence the treatment of diseases or conditions mediatedby those enzymes, such as cancer and rheumatoid arthritis. That priorart does not disclose or imply that N-formyl hydroxylamine derivativeshave antibacterial activity.

[0015] In addition to these, US-A-4,738,803 (Roques et al.) alsodiscloses N-formyl hydroxylamine derivatives, however, these compoundsare disclosed as enkephalinase inhibitors and are proposed for use asantidepressants and hypotensive agents. Also, WO 97/38705 (Bristol-MyersSquibb) discloses certain N-formyl hydroxylamine derivatives asenkephalinase and angiotensin converting enzyme inhibitors. This priorart does not disclose or imply that N-formyl hydroxylamine derivativeshave antibacterial activity either.

DETAILED DESCRIPTION OF THE INVENTION

[0016] According to the first aspect of the present invention there isprovided the use of a compound of formula (I) or a pharmaceutically orveterinarily acceptable salt thereof in the preparation of anantibacterial composition:

[0017] wherein:

[0018] R₁ represents hydrogen, or C₁-C₆ alkyl or C₁-C₆ alkyl substitutedby one or more halogen atoms;

[0019] R₂ represents a group R₁₀—(X)_(n)—(ALK)_(m)— wherein

[0020] R₁₀ represents hydrogen, or a C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, cycloalkyl, aryl, or heterocyclyl group, any of which may beunsubstituted or substituted by (C₁-C₆)alkyl, (C₁-C₆)alkoxy, hydroxy,mercapto, (C₁-C₆)alkylthio, amino, halo (including fluoro, chloro, bromoand iodo), trifluoromethyl, cyano, nitro, —COOH, —CONH₂, —COOR^(A),—NHCOR^(A), —CONH^(A), —NH^(A), —NR^(A)R^(B), or —CONR^(A)R^(B) whereinR^(A) and R^(B) are independently a (C₁-C₆)alkyl group, and

[0021] ALK represents a straight or branched divalent C₁-C₆ alkylene,C₂-C₆ alkenylene, or C₂-C₆ alkynylene radical, and may be interrupted byone or more non-adjacent —NH—, —O—or —S—linkages,

[0022] X represents —NH—, —O—or —S—, and

[0023] m and n are independently 0 or 1; and

[0024] A represents (i) a group of formula (IA), (IB), (IC) or (ID)

[0025] wherein:

[0026] R₃ represents hydrogen and R₄ represents the side chain of anatural or non-natural alpha amino acid or R₃ and R₄ when taken togetherwith the nitrogen and carbon atoms to which they are respectivelyattached form an optionally substituted saturated heterocyclic ring of 5to 8 atoms which ring is optionally fused to a carbocyclic or secondheterocyclic ring,

[0027] R₅ and R₆, independently represent hydrogen, or optionallysubstituted C₁-C₈ alkyl, cycloalkyl, aryl, aryl(C₁-C₆ alkyl),heterocyclic, or heterocyclic(C₁-C₆ alkyl), or R₅ and R₆ when takentogether with the nitrogen atom to which they are attached form anoptionally substituted saturated heterocyclic ring of 3 to 8 atoms whichring is optionally fused to a carbocyclic or second heterocyclic ring,and

[0028] R₇ represents hydrogen, C₁-C₆ alkyl, or an acyl group.

[0029] In another aspect, the invention provides a method for thetreatment of bacterial infections in humans and non-human mammals, whichcomprises administering to a subject suffering such infection anantibacterially effective dose of a compound of formula (I) as definedabove.

[0030] In a further aspect of the invention there is provided a methodfor the treatment of bacterial contamination by applying anantibacterially effective amount of a compound of formula (I) as definedabove to the site of contamination.

[0031] The compounds of formula (I) as defined above may be used ascomponent(s) of antibacterial cleaning or disinfecting materials.

[0032] According to a preferred embodiment, the various aspects of theinvention can be applied against vancomycin-, quinolone- and“β-lactam”-resistant bacteria and the infections they cause.

[0033] On the hypothesis that the compounds (I) act by inhibition ofintracellular PDF, the most potent antibacterial effect may be achievedby using compounds which efficiently pass through the bacterial cellwall. Thus, compounds which are highly active as inhibitors of PDF invitro and which penetrate bacterial cells are preferred for use inaccordance with the invention. It is to be expected that theantibacterial potency of compounds which are potent inhibitors of thePDF enzyme in vitro, but are poorly cell penetrant, may be improved bytheir use in the form of a prodrug, ie a structurally modified analoguewhich is converted to the parent molecule of formula (1), for example byenzymic action, after it has passed through the bacterial cell wall.

[0034] The invention also provides novel compounds of formula (I) above,or pharmaceutically or veterinarily acceptable salts thereof, wherein:

[0035] R₁ represents hydrogen, C₁-C₆ alkyl or C₁-C₆ alkyl substituted byone or more halogen atoms;

[0036] R₂ represents a group R₁₀—(ALK)_(m)— wherein

[0037] R₁₀ represents hydrogen, or a C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, a cycloalkyl, aryl, or heterocyclyl group, any of which may beunsubstituted or substituted by (C₁-C₆)alkyl, (C₁-C₆)alkoxy, hydroxy,mercapto, (C₁-C₆)alkylthio, amino, halo (including fluoro, chloro, bromoand iodo), trifluoromethyl, nitro, —COOH, —CONH₂, —COOR^(A), —NHCOR^(A),—CONH^(A), —NH^(A), —NR^(A)R^(B), or —CONR^(A)R^(B) wherein R^(A) andR^(B) are independently a (C₁-C₆)alkyl group,

[0038] ALK represents a straight or branched divalent C₁-C₆ alkylene,C₂-C₆ alkenylene, C₂-C₆ alkynylene radical, and may be interrupted byone or more non-adjacent —NH—, —O—or —S—linkages, and

[0039] m represents 0 or 1;

[0040] A represents a group of formula (IA), (IB), (IC) or (ID) abovewherein:

[0041] R₃ represents hydrogen and R₄ represents the side chain of anatural or non-natural alpha amino acid or R₃ and R₄ when taken togetherwith the nitrogen and carbon atoms to which they are respectivelyattached form an optionally substituted saturated heterocyclic ring of 5to 8 atoms which ring is optionally fused to a carbocyclic or secondheterocyclic ring,

[0042] R₅ and R₆, independently represent hydrogen, or optionallysubstituted C₁-C₈ alkyl, cycloalkyl, aryl(C₁-C₆ alkyl), non-aromaticheterocyclic, or heterocyclic(C₁-C₆ alkyl), or R₅ and R₆ when takentogether with the nitrogen atom to which they are attached form anoptionally substituted saturated heterocyclic ring of 3 to 8 atoms whichring is optionally fused to a carbocyclic or second heterocyclic ring,and

[0043] R₇ represents hydrogen, C₁-C₆ alkyl, or an acyl group.

[0044] PROVIDED THAT

[0045] (i) when A is a group of formula (IA) or (IB) and R₂ is C₂-C₅alkyl then R₄ is not the side chain of a natural alpha amino acid or theside chain of a natural alpha-amino acid in which any functionalsubstituents are protected, any amino groups are acylated, and anycarboxyl groups are esterified;

[0046] (ii) when A is a group of formula (IA) or (IB) then R₄ is not abicyclicarylmethyl group; and

[0047] (iii) when A is a group of formula (IA) and R₂ iscyclopropylmethyl, cyclobutylmethyl or cyclopentylmethyl and one of R₅and R₆ is hydrogen, then R₄ is not tert-butyl.

[0048] As used herein the term “(C₁-C₆)alkyl” means a straight orbranched chain alkyl moiety having from 1 to 6 carbon atoms, includingfor example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, t-butyl, n-pentyl and n-hexyl.

[0049] As used herein the term “divalent (C₁-C₆)alkylene radical” meansa saturated hydrocarbon chain having from 1 to 6 carbon atoms and twounsatisfied valencies.

[0050] As used herein the term “(C₂-C₆)alkenyl” means a straight orbranched chain alkenyl moiety having from 2 to 6 carbon atoms having atleast one double bond of either E or Z stereochemistry where applicable.The term includes, for example, vinyl, allyl, 1- and 2-butenyl and2-methyl-2-propenyl.

[0051] As used herein the term “divalent (C₂-C₆)alkenylene radical”means a hydrocarbon chain having from 2 to 6 carbon atoms, at least onedouble bond, and two unsatisfied valencies.

[0052] As used herein the term “C₂-C₆ alkynyl” refers to straight chainor branched chain hydrocarbon groups having from two to six carbon atomsand having in addition one triple bond. This term would include forexample, ethynyl, 1-propynyl, 1- and 2-butynyl, 2-methyl-2-propynyl,2-pentynyl, 3-pentynyl, 4-pentynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl and5-hexynyl.

[0053] As used herein the term “divalent (C₂-C₆)alkynylene radical”means a hydrocarbon chain having from 2 to 6 carbon atoms, at least onetriple bond, and two unsatisfied valencies.

[0054] As used herein the term “cycloalkyl” means a saturated alicyclicmoiety having from 3-8 carbon atoms and includes, for example,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl andcyclooctyl.

[0055] As used herein the term “cycloalkenyl” means an unsaturatedalicyclic moiety having from 3-8 carbon atoms and includes, for example,cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyland cyclooctenyl. In the case of cycloalkenyl rings of from 5-8 carbonatoms, the ring may contain more than one double bond.

[0056] As used herein the term “aryl” refers to a mono-, bi- ortri-cyclic carbocyclic aromatic group, and to groups consisting of twocovalently linked monocyclic carbocyclic aromatic groups. Illustrativeof such groups are phenyl, biphenyl and napthyl.

[0057] As used herein the term “heteroaryl” refers to a 5- or 6-membered aromatic ring containing one or more heteroatoms, andoptionally fused to a benzyl or pyridyl ring; and to groups consistingof two covalently linked 5- or 6- membered aromatic rings eachcontaining one or more heteroatoms; and to groups consisting of amonocyclic carbocyclic aromatic group covalently linked to a 5- or 6-membered aromatic rings containing one or more heteroatoms;.Illustrative of such groups are thienyl, furyl, pyrrolyl, imidazolyl,benzimidazolyl, thiazolyl, pyrazolyl, isoxazolyl, isothiazolyl,triazolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyridazinyl,pyrimidinyl, pyrazinyl, triazinyl, 4-([1,2,3]-thiadiazoly-4-yl)phenyland 5-isoxazol-3-ylthienyl.

[0058] As used herein the unqualified term “heterocyclyl” or“heterocyclic” includes “heteroaryl” as defined above, and in particularmeans a 5-7 membered aromatic or non-aromatic heterocyclic ringcontaining one or more heteroatoms selected from S, N and 0, andoptionally fused to a benzene ring, including for example, pyrrolyl,furyl, thienyl, piperidinyl, imidazolyl, oxazolyl, thiazolyl,thiadiazolyl, pyrazolyl, pyridinyl, pyrrolidinyl, pyrimidinyl,morpholinyl, piperazinyl, indolyl, benzimidazolyl, maleimido,succinimido, phthalimido and 1,3-dioxo-1,3-dihydro-isoindol-2-yl groups.

[0059] As used herein the term “acyl” means a group R₂₀C(O)— where R₂₀is (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₃-C₇)cycloalkyl, phenyl,heterocyclyl, phenyl(C₁-C₆)alkyl, heterocyclyl(C₁-C₆)alkyl,(C₃-C₇)cycloalkyl(C₁-C₆)alkyl, phenyl(C₂-C₆)alkenyl,heterocyclyl(C₂-C₆)alkenyl, (C₃-C₇)cycloalkyl(C₂-C₆)alkenyl, any ofwhich R₂₀ groups may be substituted.

[0060] As used herein, the term “bicyclicarylmethyl” means (i) a methylgroup substituted by a monocyclic aryl or heteroaryl group which in turnis substituted by a monocyclic aryl or heteroaryl group, or (ii) amethyl group substituted by a monocyclic aryl or heteroaryl group towhich is fused a second monocyclic aryl or heteroaryl group; andincludes both unsubstituted and substituted bicyclicarylmethyl. Examplesof such bicyclicarylmethyl groups include naphthyl, indolyl, quinolyland isoquinolyl.

[0061] Unless otherwise specified in the context in which it occurs, theterm “substituted” as applied to any moiety herein means substitutedwith up to four substituents, each of which independently may be(C₁-C₆)alkyl, benzyl, (C₁-C₆)alkoxy, phenoxy, hydroxy, mercapto,(C₁-C₆)alkylthio, amino, halo (including fluoro, chloro, bromo andiodo), trifluoromethyl, nitro, —COOH, —CONH₂, —COR^(A), —COOR^(A),—NHCOR^(A), —CONH^(A), —NH^(A), —NR^(A)R^(B), or —CONR^(A)R^(B) whereinR^(A) and R^(B) are independently a (C₁-C₆)alkyl group. In the casewhere “substituted” means benzyl, the phenyl ring thereof may itself besubstituted with any of the foregoing, except benzyl.

[0062] As used herein the terms “side chain of a natural alpha-aminoacid” and “side chain of a non-natural alpha-amino acid” mean the groupRx in respectively a natural and non-natural amino acid of formulaNH₂—CH(R^(x))—COOH.

[0063] Examples of side chains of natural alpha amino acids includethose of alanine, arginine, asparagine, aspartic acid, cysteine,cystine, glutamic acid, histidine, 5-hydroxylysine, 4-hydroxyproline,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, valine, α-aminoadipic acid,α-amino-n-butyric acid, 3,4-dihydroxyphenylalanine, homoserine,α-methylserine, ornithine, pipecolic acid, and thyroxine.

[0064] In natural alpha-amino acid side chains which contain functionalsubstituents, for example amino, carboxyl, hydroxy, mercapto, guanidyl,imidazolyl, or indolyl groups as in arginine, lysine, glutamic acid,aspartic acid, tryptophan, histidine, serine, threonine, tyrosine, andcysteine, such functional substituents may optionally be protected.

[0065] Likewise, in the side chains of non-natural alpha amino acidswhich contain functional substituents, for example amino, carboxyl,hydroxy, mercapto, guanidyl, imidazolyl, or indolyl groups, suchfunctional substituents may optionally be protected.

[0066] The term “protected” when used in relation to a functionalsubstituent in a side chain of a natural or non-natural alpha-amino acidmeans a derivative of such a substituent which is substantiallynon-functional. The widely used handbook by T. W. Greene and P. G. Wuts“Protective Groups in Organic Synthesis” Second Edition, Wiley, NewYork, 1991 reviews the subject. For example, carboxyl groups may beesterified (for example as a C₁-C₆ alkyl ester), amino groups may beconverted to amides (for example as a NHCOC₁-C₆ alkyl amide) orcarbamates (for example as an NHC(═O)OC₁-C₆ alkyl or NHC(═O)OCH₂Phcarbamate), hydroxyl groups may be converted to ethers (for example anOC₁-C₆ alkyl or a O(C₁-C₆ alkyl)phenyl ether) or esters (for example aOC(═O)C₁-C₆ alkyl ester) and thiol groups may be converted to thioethers(for example a tert-butyl or benzyl thioether) or thioesters (forexample a SC(═O)C₁-C₆ alkyl thioester).

[0067] There are several actual or potential chiral centres in thecompounds according to the invention because of the presence ofasymmetric carbon atoms. The presence of several asymmetric carbon atomsgives rise to a number of diastereoisomers with R or S stereochemistryat each chiral centre. The invention includes all such diastereoisomersand mixtures thereof. Currently, the preferred stereoconfiguration ofthe carbon atom carrying the R₂ group is R; that of the carbon atomcarrying the R₄ group (when asymmetric) is S; and that of the carbonatom carrying the R₁ group (when asymmetric) is R.

[0068] In the compounds of formula (I) as defined above for useaccording to the invention, and in the novel compounds of the inventionof formula (II) as defined above (but subject to the provisos therein):

[0069] R₁ may be, for example, hydrogen, methyl, or trifluoromethyl.Hydrogen is currently preferred.

[0070] R₂ may be, for example:

[0071] optionally substituted C₁-C₈ alkyl, C₃-C₆ alkenyl, C₃-C₆ alkynylor cycloalkyl;

[0072] phenyl(C₁-C₆ alkyl)-, phenyl(C₃-C₆ alkenyl)- or phenyl(C₃-C₆alkynyl)-optionally substituted in the phenyl ring;

[0073] cycloalkyl(C₁-C₆ alkyl)-, cycloalkyl(C₃-C₆ alkenyl)- orcycloalkyl(C₃-C₆ alkynyl)-optionally substituted in the cycloalkyl ring;

[0074] heterocyclyl(C₁-C₆ alkyl)-, heterocyclyl(C₃-C₆ alkenyl)- orheterocyclyl(C₃-C₆ alkynyl)- optionally substituted in the heterocyclylring; or CH₃(CH₂)pO(CH₂)q- or CH₃(CH₂)pS(CH₂)q-, wherein p is 0, 1, 2 or3 and q is 1, 2 or 3.

[0075] Specific examples of R₂ groups include

[0076] methyl, ethyl, n- and iso-propyl, n- and iso-butyl, n-pentyl,iso-pentyl 3-methylbut-1-yl, n-hexyl, n-heptyl, n-acetyl, n-octyl,methylsulfanylethyl, ethylsulfanylmethyl, 2-methoxyethyl, 2-ethoxyethyl,2-ethoxymethyl, 3-hydroxypropyl, allyl, 3-phenylprop-3-en-1-yl,prop-2-yn-1-yl, 3-phenylprop-2-yn-1-yl,3-(2-chlorophenyl)prop-2-yn-1-yl, but-2-yn-1-yl, cyclopentyl,cyclohexyl, cyclopentylmethyl, cyclopentylethyl, cyclopentylpropyl,cyclohexylmethyl, cyclohexylethyl, cyclohexylpropyl, furan-2-ylmethyl,furan-3-methyl, tetrahydrofuran-2-ylmethyl, tetrahydrofuran-2-ylmethyl,piperidinylmethyl, phenylpropyl, 4-chlorophenylpropyl,4-methylphenylpropyl, 4-methoxyphenylpropyl, benzyl, 4-chlorobenzyl,4-methylbenzyl, and 4-methoxybenzyl.

[0077] Presently preferred groups at R₂ are n-propyl, n-butyl, n-pentyl,benzyl and cyclopentylmethyl.

[0078] In the case of R₃, hydrogen is presently preferred.

[0079] R₄ may be, for example

[0080] the characterising group of a natural α amino acid, for examplebenzyl, or 4-methoxyphenylmethyl, in which any functional group may beprotected, any amino group may be acylated and any carboxyl grouppresent may be amidated; or  a group —[Alk]_(n)R₉ where Alk is a(C₁-C₆)alkylene or (C₂-C₆)alkenylene group optionally interrupted by oneor more —O—, or —S—atoms or —N(R₁₂)— groups [where R₁₂ is a hydrogenatom or a (C₁-C₆)alkyl group], n is 0 or 1, and R₉ is hydrogen or anoptionally substituted phenyl, aryl, heterocyclyl, cycloalkyl orcycloalkenyl group or (only when n is 1) R₉ may additionally be hydroxy,mercapto, (C₁-C₆)alkylthio, amino, halo, trifluoromethyl, nitro, —COOH,—CONH₂, —COOR^(A), —NHCOR^(A), —CONH^(A), -NH^(A), NR^(A)R^(B), orCONR^(A)R^(B)wherein R^(A) and R^(B) are independently a (C₁-C₆)alkylgroup; or

[0081] a benzyl group substituted in the phenyl ring by a group offormula —OCH₂COR₈ where R₈ is hydroxyl, amino, (C₁-C₆)alkoxy,phenyl(C₁-C₆)alkoxy, (C₁-C₆)alkylamino, di((C₁-C₆)alkyl)amino,phenyl(C₁-C₆)alkylamino; or

[0082] a heterocyclic(C₁-C₆)alkyl group, either being unsubstituted ormono- or di-substituted in the heterocyclic ring with halo, nitro,carboxy, (C₁-C₆)alkoxy, cyano, (C₁-C₆)alkanoyl, trifluoromethyl(C₁-C₆)alkyl, hydroxy, formyl, amino, (C₁-C₆)alkylamino,di-(C₁-C₆)alkylamino, mercapto, (C₁-C₆)alkylthio, hydroxy(C₁-C₆)alkyl,mercapto(C₁-C₆)alkyl or (C₁-C₆)alkylphenylmethyl; or

[0083] a group —CR_(a)R_(b)R_(c) in which:

[0084] each of R_(a), R_(b) and R_(c) is independently hydrogen,(C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, phenyl(C₁-C₆)alkyl,(C₃-C₈)cycloalkyl; or

[0085] R_(c) is hydrogen and R_(a) and R_(b) are independently phenyl orheteroaryl such as pyridyl; or

[0086] R_(c) is hydrogen, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,phenyl(C₁-C₆)alkyl, or (C₃-C₈)cycloalkyl, and Ra and Rb together withthe carbon atom to which they are attached form a 3 to 8 memberedcycloalkyl or a 5- to 6-membered heterocyclic ring; or

[0087] R_(a), R_(b) and R_(c) together with the carbon atom to whichthey are attached form a tricyclic ring (for example adamantyl); or

[0088] R_(a) and R_(b) are each independently (C₁-C₆)alkyl,(C₂-C₆)alkenyl, (C₂-C₆)alkynyl, phenyl(C₁-C₆)alkyl, or a group asdefined for R_(c) below other than hydrogen, or R_(a) and R_(b) togetherwith the carbon atom to which they are attached form a cycloalkyl orheterocyclic ring, and R_(c) is hydrogen, —OH, —SH, halogen, —CN, —CO₂H,(C₁-C₄)perfluoroalkyl, —CH₂OH, —CO₂(C₁-C₆)alkyl, —O(C₁-C₆)alkyl,—O(C₂-C₆)alkenyl, —S(C₁-C₆)alkyl, —SO(C₁-C₆)alkyl, —SO₂(C₁-C₆) alkyl,—S(C₂-C₆)alkenyl, —SO(C₂-C₆)alkenyl, —SO₂(C₂-C₆)alkenyl or a group —Q—Wwherein Q represents a bond or —O—, —S—, —SO— or —SO₂— and W representsa phenyl, phenylalkyl, (C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkylalkyl,(C₄-C₈)cycloalkenyl, (C₄-C₈)cycloalkenylalkyl, heteroaryl orheteroarylalkyl group, which group W may optionally be substituted byone or more substituents independently selected from, hydroxyl, halogen,—CN, —CO₂H, —CO₂(C₁-C₆)alkyl, —CONH₂, —CONH(C₁-C₆)alkyl,—CONH(C₁-C₆alkyl)₂, —CHO, —CH₂OH, (C₁-C₄)perfluoroalkyl, —O(C₁-C₆)alkyl,—S(C₁-C₆)alkyl, —SO(C₁-C₆)alkyl, —SO₂(C₁-C₆)alkyl, -NO₂, —NH₂,—NH(C₁-C₆)alkyl, —N((C₁-C₆)alkyl)₂, —NHCO(C₁-C₆)alkyl, (C₁-C₆)alkyl,(C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₃-C₈)cycloalkyl, (C₄-C₈)cycloalkenyl,phenyl or benzyl.

[0089] Examples of particular R₄ groups include methyl, ethyl, benzyl,4-chlorobenzyl, 4-hydroxybenzyl, phenyl, cyclohexyl, cyclohexylmethyl,pyridin-3-yimethyl, tert-butoxymethyl, naphthylmethyl, iso-butyl,sec-butyl, tert-butyl, 1-benzylthio-1-methylethyl,1-methylthio-1-methylethyl, 1-mercapto-1-methylethyl,1-methoxy-1-methylethyl, 1-hydroxy-1-methylethyl,1-fluoro-1-methylethyl, hydroxymethyl, 2-hydroxethyl, 2-carboxyethyl,2-methylcarbamoylethyl, 2-carbamoylethyl, and 4-aminobutyl. Presentlypreferred R₄ groups include tert-butyl, iso-butyl, benzyl and methyl.

[0090] R₃ and R₄ when taken together with the nitrogen and carbon atomsto which they are respectively attached may form an optionallysubstituted saturated heterocyclic ring of 5 to 8 atoms. For example, R₃and R₄ may form a bridge between the nitrogen and carbon atoms to whichthey are attached, said bridge being represented by the divalent radical—(CH₂)₃₋₆—, or —(CH₂)_(r)—O—(CH₂)_(s)—, or —(CH₂)_(r)—S—(CH₂)_(s)—,wherein r and s are each independently 1, 2 or 3 with the proviso thatr+s =2, 3, 4, or 5.

[0091] R₅ and R₆ may independently be, for example, hydrogen, methyl,ethyl, tert-butyl, cyclopentyl, cyclohexyl,1,1,3,3-tetramethylbutyl,benzyl, or 2-hydroxyethyl; or R₅ and R₆ whentaken together with the nitrogen atom to which they are attached mayform a saturated 5- to 8-membered monocyclic N-heterocyclic ring whichis attached via the N atom and which optionally contains —N(R₁₁)—wherein R₁₁ is hydrogen or C₁-C₆ alkyl, benzyl, acyl, or an aminoprotecting group, O, S, SO or SO₂ as a ring member, and/or is optionallysubstituted on one or more C atoms by hydroxy, C₁-C₆ alkyl,hydroxy(C₁-C₆ alkyl)-, C₁-C₆ alkoxy, oxo, ketalised oxo, amino,mono(C₁-C₆ alkyl)amino, di(C₁-C₆ alkyl)amino, carboxy, C₁-C₆alkoxycarbonyl, hydroxymethyl, C₁-C₆ alkoxymethyl, carbamoyl, mono(C₁-C₆alkyl)carbamoyl, di(C₁-C₆ alkyl)carbamoyl, or hydroxyimino.

[0092] Examples of such rings are substituted or unsubstituted1-pyrrolidinyl, piperidin-1-yl, 1-piperazinyl, hexahydro-1-pyridazinyl,morpholin-4-yl, tetrahydro-1,4-thiazin-4-yl, tetrahydro-1,4-thiazin-4-yl1-oxide, tetrahydro-1,4-thiazin-4-yl 1,1-dioxide, hexahydroazipino, oroctahydroazocino. Substituted examples of the foregoing are2-(methylcarbamoyl)-1-pyrrolidinyl, 2-(hydroxymethyl)-1-pyrrolidinyl,4-hydroxypiperidino, 2-(methylcarbamoyl)piperidino,4-hydroxyiminopiperidino, 4-methoxypiperidino, 4-methylpiperidin-1yl,4-benzylpiperidin-1-yl, 4-acetylpiperidin-1-yl,4-methyl-1-piperazinyl,4-phenyl-1-piperazinyl, 1,4-dioxa-8-azaspiro[4,5]decan-8-yl,hexahydro-3-(methylcarbamoyl)-2-pyridazinyl, andhexahydro-1-(benzyloxycarbonyl)-2-pyridazinyl,decahydroisoquinolin-2-yl, and 1,2,3,4-tetrahydroisoquinolin-2-yl.

[0093] When A is a group of formula (IA), it is currently preferred thatR₅ be methyl or hydrogen, and R₆ be methyl.

[0094] R₇ may be, for example, hydrogen, or a group R₂₀C(O)— where R₂₀is a (C₁-C₆)alkyl group such as methyl or ethyl.

[0095] Specific examples of compounds useful as antibacterial agents inaccordance with the invention include those of the specific Examplesherein. Preferred novel compounds of the invention include

[0096] 2R (orS)-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-dimethylcarbamoyl-ethyl)-amide and

[0097] 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-3-cyclopentyl-propionicacid (1S-dimethyl-carbamoyl-2,2-dimethyl-propyl)-amide

[0098] and their pharmaceutically and veterinarily acceptable salts.

[0099] Compounds with which the invention is concerned the invention maybe prepared by deprotecting an 0-protected N-formyl-N-hydroxyaminocompound of formula (II):

[0100] in which R₁, R₂, and A are as defined in general formula (I) andR₂₅ is a hydroxy protecting group removable to leave a hydroxy group byhydrogenolysis or hydrolysis. Benzyl is a preferred R₂₅ group forremoval by hydrogenolysis, and tert-butyl and tetrahydropyranyl arepreferred groups for removal by acid hydrolysis.

[0101] Compounds of formula (II) wherein A is a group of formula (IA),(IB), (IC) or (ID) may be prepared by causing an acid of formula (III)or an activated derivative thereof to react with an amine of formula(IVA), (IVB), (IVC) or (IVD) respectively

[0102] wherein R₁ R₂, R₃, R₄, R₅, R₆ and R₇ are as defined in generalformula (I) except that the —OH group in (IVB) and any substituents inR₁ R₂, R₃, R₄, R₅, R₆ and R₇ which are potentially reactive in thecoupling reaction may themselves be protected from such reaction, andR₂₅ is as defined in relation to formula (II) above, and optionallyremoving protecting groups from the —OH group in (IVB) and R₁ R₂, R₃,R₄, R₅, R₆ and R₇.

[0103] Compounds of formula (III) may be prepared by N-formylation, forexample using acetic anhydride and formic acid, or1-formylbenzotriazole, of compounds of formula (V)

[0104] wherein R₁, R₂ and R₂₅ are as defined in relation to formula (II)and X is either a chiral auxiliary or an OR₂₆ group wherein R₂₆ ishydrogen or a hydroxy protecting group. In the case where X is an OR₂₆group or a chiral auxiliary the hydroxy protecting group or auxiliary isremoved after the formylation step to provide the compound of formula(V). Suitable chiral auxiliaries include substituted oxazolidinoneswhich may be removed by hydrolysis in the presence of base.

[0105] In an alternative procedure compounds of general formula (II) maybe prepared by N-formylation, for example using acetic anhydride andformic acid, or 1-formylbenzotriazole, of compounds of formula (VI)

[0106] wherein R₁, R₂, R₂₅ and A are as defined in relation to formula(II).

[0107] Compounds of formula (VI) wherein A is a group of formula (IA),(IB), (IC) or (ID) may be prepared by causing an acid of general formula(VII) or an activated derivative thereof

[0108] wherein R₁, R₂ and R₂₅ are as defined in relation to formula (II)to react with an amine of formula (IVA), (IVB), (IVC) or (IVD)respectively as defined above.

[0109] Alternatively compounds of general formula (VI) may be preparedby reduction of an oxime of general formula (VIII).

[0110] Reducing agents include certain metal hydrides (e.g. sodiumcyanoborohydride in acetic acid, triethylsilane or borane/pyridine) andhydrogen in the presence of a suitable catalyst.

[0111] In an alternative procedure compounds of general formula (II)wherein R₁ and R₂ are as defined in general formula (I), R₂₅ is ahydroxy protecting group as defined above and A is a group of formula(IA) wherein R₃, R₄, R₅ are as defined in general formula (IA) and R₆ ishydrogen may be prepared by a 4-component Ugi reaction of carboxylicacid of general formula (III) as defined above, an amine of formula(IX), an aldehyde of formula (X) and an isonitrile of formula (XI)

R₃—NH₂  (IX)

R—CHO  (X)

R₅—CN  (XI)

[0112] wherein R₃, R₄ and R₅ are as defined above.

[0113] A compound of general formula (V) may be prepared by reduction ofan oxime of general formula (XI)

[0114] wherein R₁, R₂, and R₂₅ are as defined above, and X is either anOR₂₆ group as defined above or a chiral auxiliary. Reducing agentsinclude certain metal hydrides (eg sodium cyanoborohydride in aceticacid, triethylsilane or borane/pyridine) and hydrogen in the presence ofa suitable catalyst. Following the reduction when the group X is achiral auxiliary it may be optionally converted to a OR₂₆ group.

[0115] A compound of general formula (Xl) can be prepared by reaction ofa P-keto carbonyl compound of general formula (XII)

[0116] wherein R₁, R₂, and X are as defined above, with an 0-protectedhydroxylamine.

[0117] β-keto carbonyl compounds (XII) may be prepared in racemic formby formylation or acylation of a carbonyl compound of general formula(XIII)

[0118] wherein R₂ and X are as defined above, with a compound of generalformula (XIV)

[0119] wherein R₁ is as defined above and Z is a leaving group such ashalogen or alkoxy, in the presence of a base.

[0120] Another method for the preparation of a compound of generalformula (V) is by Michael addition of a hydroxylamine derivative to α,β-unsaturated carbonyl compounds of general formula (XV)

[0121] wherein R₁, R₂, and X are as defined above. Following the Michaeladdition reaction, when the group X is a chiral auxiliary it may beoptionally converted to a OR₂₆ group. The α,β-unsaturated carbonylcompounds (XV) may be prepared by standard methods.

[0122] Salts of the compounds of the invention include physiologicallyacceptable acid addition salts for example hydrochlorides,hydrobromides, sulphates, methane sulphonates, p-toluenesulphonates,phosphates, acetates, citrates, succinates, lactates, tartrates,fumarates and maleates. Salts may also be formed with bases, for examplesodium, potassium, magnesium, and calcium salts.

[0123] Compositions with which the invention is concerned may beprepared for administration by any route consistent with thepharmacokinetic properties of the active ingredient(s).

[0124] Orally administrable compositions may be in the form of tablets,capsules, powders, granules, lozenges, liquid or gel preparations, suchas oral, topical, or sterile parenteral solutions or suspensions.Tablets and capsules for oral administration may be in unit dosepresentation form, and may contain conventional excipients such asbinding agents, for example syrup, acacia, gelatin, sorbitol,tragacanth, or polyvinyl-pyrrolidone; fillers for example lactose,sugar, maize-starch, calcium phosphate, sorbitol or glycine; tablettinglubricant, for example magnesium stearate, talc, polyethylene glycol orsilica; disintegrants for example potato starch, or acceptable wettingagents such as sodium lauryl sulphate. The tablets may be coatedaccording to methods well known in normal pharmaceutical practice. Oralliquid preparations may be in the form of, for example, aqueous or oilysuspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives such as suspending agents, for example sorbitol,syrup, methyl cellulose, glucose syrup, gelatin hydrogenated ediblefats; emulsifying agents, for example lecithin, sorbitan monooleate, oracacia; non-aqueous vehicles (which may include edible oils), forexample almond oil, fractionated coconut oil, oily esters such asglycerine, propylene glycol, or ethyl alcohol; preservatives, forexample methyl or propyl p-hydroxybenzoate or sorbic acid, and ifdesired conventional flavouring or colouring agents.

[0125] For topical application to the skin, the active ingredient(s) maybe made up into a cream, lotion or ointment. Cream or ointmentformulations which may be used for the drug are conventionalformulations well known in the art, for example as described in standardtextbooks of pharmaceutics such as the British Pharmacopoeia.

[0126] The active ingredient(s) may also be administered parenterally ina sterile medium. Depending on the vehicle and concentration used, thedrug can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as a local anaesthetic, preservative andbuffering agents can be dissolved in the vehicle. Intra-venous infusionis another route of administration for the compounds used in accordancewith the invention.

[0127] Safe and effective dosages for different classes of patient andfor different disease states will be determined by clinical trial as isrequired in the art. It will be understood that the specific dose levelfor any particular patient will depend upon a variety of factorsincluding the activity of the specific compound employed, the age, bodyweight, general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination and the severity ofthe particular disease undergoing therapy.

[0128] The finding that compounds with PDF inhibitory activity caninhibit or prevent bacterial growth, opens up a novel approach foridentifying new antibacterial agents by screening test compounds foractivity as inhibitors of PDF in vitro, followed by confirmation oftheir antibacterial ability using bacterial growth inhibition studies.This finding also makes available (i) the use of compounds with PDFinhibitory activity as antibacterial agents, and (ii) a method for thetreatment of bacterial infection or contamination by applying oradministering a compound which inhibits the activity of bacterial PDF.

[0129] According to a further aspect of the invention therefore, thereis provided a method for the identification of antibacterial compounds,comprising screening test compounds for their ability to inhibit PDF invitro, selecting those compounds which exhibit said ability and testingthese for their ability to inhibit bacterial growth. The ability toinhibit bacterial growth can be performed using classical plate or brothculture bacterial growth inhibition studies, such as those performed inthe Biological Examples herein.

[0130] A suitable in vitro PDF inhibition screen may comprise mixingtogether PDF, a PDF substrate, preferably, labelled with a detectablemarker, and the test compound and assessing after a suitable length oftime whether or not the presence of the test compound inhibits theability of PDF to deformylate the substrate.

[0131] In a preferred embodiment, the cleaved substrate is detected witha fluorogenic marker such as fluorescamine. On removal of the formylgroup from the N-terminal methionine of the PDF substrate, the freeamino group reacts with fluorescamine generating a fluorescent product.

[0132] An alternative screen involves assessing whether a proteinexpressed by bacteria that express endogenous (or recombinantlyexpressed) PDF, when grown in the presence of a test compound, yieldssuitable substrate for N-terminal sequencing, or yields a lesser amountof substrate, than protein expressed from the same bacteria grown in theabsence of the test compound. Such a method could be based on that usedin the Biological Examples herein.

[0133] The person skilled in the art will be able to develop, withoutinventive input, alternative methods for screening test compounds fortheir ability to inhibit bacterial PDF.

[0134] The natural antibiotic actinonin (see for example J. C. S PerkinI, 1975, 819) is a hydroxamic acid derivative of Structure (A):

[0135] In addition to actinonin, various structural analogues ofactinonin have also been shown to have antibacterial activity (see forexample Broughton et al. (Devlin et al. Journal of the Chemical Society.Perkin Transactions 1 (9):830-841, 1975; Broughton et al. Journal of theChemical Society. Perkin Transactions 1 (9):857-860, 1975).

[0136] To date, however, the mechanism underlying the antibacterialactivity of actinonin has not been known. The present inventors havefound that actinonin inhibits the activity of bacterial PDF.

[0137] The matlystatin group of compounds share a number of structuralsimilarities with actinonin. Both are peptidic molecules with functionalhydroxamic acid metal binding groups (Ogita et al., J. Antibiotics.45(11):1723-1732; Tanzawa et al., J. Antibiotics. 45(11):1733-1737;Haruyama et al., J. Antibiotics. 47(12):1473-1480; Tamaki et al., J.Antibiotics. 47(12):1481-1492). The matlystatins and their closestructural analogues are characterised by the presence in the moleculeof a divalent piperazin-1,6-diyl group, i.e.

[0138] In view of their close structural similarity to actinonin, theobservation that actinonin inhibits PDF implies that matlystatincompounds may also inhibit PDF.

[0139] According to a further aspect of the present invention there isprovided the use of a compound which inhibits the activity of bacterialPDF, in the preparation of an antibacterial composition or agent,provided that (i) the compound is not of formula (XI)

RCO—CH(W)—NH—CO—CH(Y)—CH₂—CO—NH—OH  (XI)

[0140] wherein,

[0141] (a) R is a cyclic amino group, W is hydrogen, methyl, isopropyl,isobutyl or benzyl, and Y is hydrogen, C₁-C₆ alkyl, phenyl, benzyl,4-chlorophenylmethyl, 4-nitrophenylmethyl, or 4-aminophenylmethyl; or,

[0142] (b) R is 2-pyridylamino or 2-thiazolylamino, W is isopropyl and Yis n-pentyl; or,

[0143] (c) R is diethylamino, W is methyl or isopropyl and Y isn-pentyl; or (ii) the compound is not one containing a divalentpiperazin-1, 6-diyl group, i.e. a group of formula (XII):

[0144] According to a further aspect of the invention there is provideda method of treating bacterial infection or contamination byadministering to a patient suffering such infection or contamination, orapplying to the site of such infection or contamination, anantibacterially effective amount of a compound which inhibits theactivity of bacterial PDF enzyme, provided that the compound is not oneprovided in the provisos in the immediately preceeding paragraph.

[0145] These provisos exclude actinonin and its antibacterially activeanalogues as disclosed in Devlin et al., Journal of the ChemicalSociety. Perkin Transactions 1 (9):830-841,1975 and Broughton et al.Journal of the Chemical Society. Perkin Transactions 1 (9):857-860,1975, and the matlystatin compounds disclosed in Ogita et al., J.Antibiotics. 45(11):1723-1732; Tanzawa et al., J. Antibiotics.45(11):1733-1737; Haruyama et al., J. Antibiotics. 47(12):1473-1480 andTamaki et al., J. Antibiotics. 47(12):1481-1492.

[0146] The following examples illustrate embodiments of the invention.L-tert-Leucine-N-methylamide and L-tert-leucine-N,N-dimethylamide andother amino acid derivatives were prepared according to establishedliterature methods.

[0147] The following abbreviations have been used throughout:

[0148] DMF N,N-Dimethylformamide

[0149] EDC N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0150] HOAt 1-Hydroxy-7-aza-benzotriazole

[0151] HOBt 1-Hydroxybenzotriazole

[0152] HPLC High performance liquid chromatography

[0153] LRMS Low resolution mass spectrometry

[0154] TLC Thin layer chromatography

[0155]¹H and ¹³C NMR spectra were recorded using a Bruker AC₂₅₀Espectrometer at 250.1 and 62.9 MHz, respectively. Mass spectra wereobtained using a Perkin Elmer Sciex API 165 spectrometer using bothpositive and negative ion modes. Infra-red spectra were recorded on aPerkin Elmer PE 1600 FTIR spectrometer.

EXAMPLE 1

[0156] 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-hexanoicacid-(2,2-dimethyl-1S-methyl-carbamoyl-propyl)-amide

[0157] The title compound was prepared according to the route outlinedin Scheme 1 and as described in detail below:

[0158] STEP A: 2-Butyl-acrylic Acid

[0159] Butylmalonic acid (25 g, 156 mmol) was dissolved in ethanol (250ml) and 37% formaldehyde solution (15.45 ml, 156 mmol) was addedfollowed by piperidine (47 ml, 624 mmol). The mixture was stirredovernight at 80° C. under a reflux condenser. The solvents were removedunder reduced pressure and the residue was diluted with 1 M hydrochloricacid and extracted with dichloromethane (3×30 ml). The combined organicextracts were washed with brine, dried over anhydrous magnesium sulfate,filtered and evaporated to afford the desired product as a yellow oil(25 g, with residual solvent). ¹H-NMR: δ (CDCl₃), 10.04 (1H, br s), 6.22(1H, s), 5.57 (1H, d, J =1.3 Hz), 2.30 (2H, t, J =6.9 Hz), 1.38 (4H, m),and 0.91 (3H, t, J =7.2 Hz).

[0160] STEP B: 2RS-(Benzyloxy-amino-methyl)-hexanoic Acid

[0161] A mixture of 2-butyl-acrylic acid (3.43 g, 27.1 mmol) andO-benzylhydroxylamine (5 g, 40.65 mmol) were heated at 80° C. overnight.The mixture was cooled to room temperature, diluted with ethyl acetate(40 ml), and washed with 1 M hydrochloric acid (3×20 ml), saturatedsodium hydrogen carbonate solution (2×20 ml) and brine (2×20 ml), driedover anhydrous magnesium sulfate, filtered and evaporated to leave thetitle compound as a white solid (2.62 g, 39%). ¹H-NMR: δ (CDCl₃), 8.05(1H, br s), 7.35 (5H, m), 5.00 (2H, m), 3.28 (2H, m), 2.98 1 H, m), 1.31(6H, m) and 0.88 (3H, t, J =5.0 Hz).

[0162] Step C: 2RS-[(Benzyloxy-formyl-amino)-methyl]-hexanoic Acid

[0163] 2RS-(Benzyloxyamino-methyl)-hexanoic acid (2.62 g, 10.51 mmol)was dissolved in formic acid (4 ml, 105 mmol) and acetic anhydride (1.9ml, 21.02 mmol) and stirred overnight at room temperature. The solutionwas diluted with ethyl acetate (40 ml), washed with water (2×20 ml),saturated sodium hydrogen carbonate solution (20 ml) and brine (20 ml),dried over anhydrous magnesium sulfate, filtered and evaporate to leavethe desired product as a yellow oil (2.9 g, 99%). ¹H-NMR: δ (CDCl₃,rotamers), 8.21 (0.5H, s), 8.14 (0.5H, s), 7.37 (5H, m), 4.98 (2H, m),3.86 (1H, m), 3.27 (0.5H, dd, J =6.0,14.0 Hz), 2.93 (0.5H, m), 2.77 (1H,m), 1.50 (2H, m), 1.30 (4H, m) and 0.88 (3H, m).

[0164] STEP D: 2RS-[(Benzyloxy-formyl-amino)-methyl]-hexanoic AcidPentafluorophenyl Ester

[0165] 2RS-[(Benzyloxy-formyl-amino)-methyl]-hexanoic acid (30.72 g, 110mmol) and penta-fluorophenol (26.31 g, 143 mmol) were dissolved indichloromethane (150 ml) and the solution was stirred and cooled in anice bath during addition of EDC (25.3 g, 131 mmol). The reaction mixturewas allowed to warm to room temperature and stirred overnight. Thesolution was washed successively with 1 M hydrochloric acid (2×50 ml),0.5M sodium carbonate (2×50 ml) and brine (50 ml), dried over anhydrousmagnesium sulfate and filtered. The filtrate was evaporated underreduced pressure and the residue was purified by flash chromatography(silica gel, dichloromethane) to afford the title compound as acolourless oil (15.0 g, 31%). ¹H-NMR: δ (CDCl₃, rotamers), 8.17 (1H, brs), 7.37 (5H, m), 4.95-4.70 (2H, br m), 4.10-3.75 (2H, br m), 3.10 (1H,br s), 1.88-1.55 (2H, m), 1.39 (4H, m) and 0.92 (3H, t, J =7.0 Hz).

[0166] STEP E: 2R (or S)-[(Benzyloxy-formyl-amino)-methyl]-hexanoicacid-(2,2-dimethyl-1-methyl-carbamoyl-propyl)-amide

[0167] 2RS-[(Benzyloxy-formyl-amino)-methyl]-hexanoic acidpentafluorophenyl ester (5 g, 11 mmol) and tert-leucine N-methylamide(1.62 g, 11 mmol) were dissolved in DMF (60 ml) and the mixture wasstirred overnight at 35° C. The solvent was removed under reducedpressure and the residue was redissolved in dichloromethane. Thesolution was washed successively with 0.5 M sodium carbonate, 1.0 Mhydrochloric acid and brine, dried over anhydrous magnesium sulfate andfiltered. The two diastereoisomeric products were separated by flashchromatography (silica gel, gradient elution with 30% to 0% hexane inethyl acetate). Diastereoisomer A (higher Rf): ¹H-NMR: δ (CDCl₃,rotamers), 8.12, 7.87 (1H, 2br s), 7.27 (5H, m), 6.26 (1H, d, J =8.7Hz), 5.78 (1H, br s), 4.91-4.60 (2H, br m), 4.15 (1H, d, J =9.2 Hz),3.75 (2H, br m), 2.79 (3H, d, J =4.8 Hz), 2.56 (1H, m), 1.60-1.35 (2H,br m), 1.24 (4H, m), 0.96 (9H, s) and 0.86 (3H, t, J =6.7 Hz).Diastereoisomer B (lower Rf): ¹H-NMR: δ (CDCl₃, rotamers), 8.16, 7.88(1H, 2br s), 7.27 (5H, m), 6.28 (1H, d, J =8.9 Hz), 5.70-5.44 (1H, brs), 4.98-4.61 (2H, br m), 4.14 (1H, d, J =9.2 Hz), 3.78-3.62 (2H, br m),2.85-2.60 (3H, br m), 2.47 (1H, m), 1.72-1.25 (6H, br m), 0.98 (9H, s)and 0.88 (3H, t, J =6.7 Hz).

[0168] STEP F: 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-hexanoicacid-(2,2-dimethyl-1S-methylcarbamoyl-propyl)-amide

[0169] 2-[(Benzyloxy-formyl-amino)-methyl]-hexanoicacid-(2,2-dimethyl-1-methylcarbamoyl-propyl)-amide (diastereoisomer A)(1.0 g, 2.5 mmol) was dissolved in a mixture of ethyl acetate (20 ml)and ethanol (1 ml) and the solution was placed under an argonatmosphere. 10% palladium on charcoal was added and a fine stream ofhydrogen gas was bubbled through the suspension. After 40 minutes TLCanalysis revealed that all the starting material had been consumedleaving a more polar, ferric chloride positive species. The system wasflushed with argon before removing the catalyst by filtration. Thefiltrate was evaporated to dryness to leave the title compound as anoff-white foam (810 mg, including residual solvent). ¹H-NMR: δ((CD₃)₂SO, rotamers), 9.81, 9.41 (1H, 2br s), 7.82-7.60 (3H, m), 4.04(1H, d, J =9.3 Hz), 3.50-3.02 (2H, m), 2.87-2.60 (1H, m), 2.41 (3H, d, J=4.5 Hz), 1.39-0.93 6H, m), 0.75 (9H, s) and 0.67 (3H, t, J =5.7 Hz).¹³C-NMR: δ ((CD₃)₂SO), 172.5, 170.2, 157.5, 59.9, 42.8, 33.3, 29.0,28.4, 28.2, 26.4, 24.8, 21.7 and 13.3. IR (KBr disc), Vmax 3309, 2959,2873,1646 and 1540 cm⁻¹.

[0170] 2-[(Benzyloxy-formyl-amino)-methyl]-hexanoicacid-(2,2-dimethyl-1-methylcarbamoyl-propyl)-amide (diastereoisomer B)(1.0 g, 2.5 mmol) was similarly deprotected to give diastereoisomer B ofthe title compound (740 mg, 97%). ¹H-NMR: δ ((CD₃)₂SO, rotamers), 9.75,9.30 (1H, 2br s), 7.81-7.42 (3H, m), 4.04 (1H, d, J =9.5 Hz), 3.53-3.02(2H, m), 2.80-2.55 (1H, m), 2.41 (3H, d, J =4.5 Hz), 1.33-0.82 (6H, m),0.72 (9H, s) and 0.67 (3H, t, J =6.7 Hz). ¹³C-NMR: δ ((CD₃)₂SO),172.6,170.4,161.7, 157.0, 59.8, 34.0, 29.4, 28.6, 26.7, 25.2, 22.1 and14.1. IR (KBr disc), Vmax 3312, 2959, 1640, 1541, 1369 and 1240 cm⁻¹.

EXAMPLE 2

[0171] 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-hexanoicacid-(2,2-dimethyl-1S-tert-butyl-carbamoyl-propyl)-amide

[0172] The title compounds were prepared by analogy with Example 1,using the L-tert-leucine-N-tert-butylamide in place ofL-tert-leucine-N-methylamide in Step E. The diastereoisomers were notseparable by flash chromatography (silica gel, ethyl acetate) at Step Eand were converted to a mixture of the desired N-formyl hydroxylaminederivatives by hydrogenolysis. White solid. ¹³C-NMR: δ ((CD₃)₂SO),172.8,172.5,170.1, 169.6,161.6,156.9, 59.9, 59.7, 51.9, 51.7, 50.2,49.6, 48.3, 43.2, 43.1, 42.7, 34.2, 34.0, 29.6, 29.3, 29.2, 28.8, 28.6,26.7, 22.2, 22.1, 20.3 and 13.9. IR (KBr), vmax 3311,2964,1639,1548,1456,1394,1364 and 1226 cm⁻¹.

EXAMPLE 3

[0173] 2R (orS)-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-methyl-2-morpholin-4-yl-2-oxo-ethyl)-amide

[0174] A solution of 2RS-[(benzyloxy-formyl-amino)-methyl]-hexanoic acidpentafluorophenyl ester (Example 1, Step D) (445 mg, 1 mmol) in DMF (5ml) was added to L alanine-N-morpholinoamide (158 mg, 1 mmol) in aboiling tube and stirred at 35° C. overnight. DMF was removed in vacuoand the residue was redissolved in dichloromethane (2 ml) and passedthrough a purification cartridge (Isolute-NH₂), eluting withdichloromethane (4 ml) in order to remove pentafluorophenol.Dichloromethane was removed under reduced pressure and the residue wasredissolved in formic acid (2 ml) and ethyl acetate (2 ml). The solutionwas then treated with 10% palladium on charcoal (200 mg) and stirred atroom temperature for 2 hours. Catalyst was removed by filtration throughcelite, washing well with methanol and solvents were removed in vacuo.Compounds were purified by reverse phase HPLC (gradient elution, 10-90%acetonitrile/water). Diastereoisomer A: ¹H-NMR; δ (CD₃OD), 8.03 (0.5H,s), 7.84 (0.5H, s), 4.75 (1H, m), 3.65 (8H, m), 3.39 (1H, m), 3.24 (1H,dd, J=4.0, 13.2 Hz), 2.84 (1H, m), 1.57 (2H, m), 1.34 (7H, m), and 0.92(3H, m). LRMS: −ve ion 328 [M−H]. Diastereoisomer B: ¹H-NMR; δ (CD₃OD),3.66 (8H, m), 3.41 (1H, dd, J=9.98, 13.1 Hz), 3.23 (1H, m), 2.90 (0.5H,m), 2.71 (0.5H, m), 1.62 (2H, m), 1.33 (7H, m), and 0.92 (3H, t, J=6.7Hz). LRMS: −ve ion 328 [M−H].

[0175] The compounds of Examples 4 to 12 were prepared by analogy withExample 3 using the appropriate amine component in place ofL-alanine-N-morpholinoamide. Where both diastereoisomers were prepared,diastereoisomer A is the faster eluting and more potent against PDF invitro. In some cases only the faster running diastereoisomer was takenthrough to the final product.

EXAMPLE 4

[0176] 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-dimethylcarbamoylethyl)-amide

[0177] Diastereoisomer A: ¹H-NMR; δ (CD₃OD), 4.72 (1H, m), 3.53 (1H, dd,J=8.9,13.0 Hz), 3.23 (1H, m), 3.14 (3H, s), 2.95 (3H, s), 2.83 (0.5H,m), 2.74 (0.5H, m), 1.57 (2H, m), 1.33 (7H, m) and 0.92 (3H, m). LRMS;+ve ion 288 [M+H], −ve ion 286 [M−H].

[0178] Diastereoisomer B: ¹ H-NMR; δ (CD₃OD), 4.74 (1H, m), 3.41 (1H,dd, J=9.9, 13.0 Hz), 3.25 (1H, dd, J=4.0, 13.1 Hz), 3.15 (3H, s), 2.97(3H, s), 2.89 (0.5H, m), 2.72 (0.5H, m), 1.53 (2H, m), 1.33 (7H, m) and0.93 (3H, t, J=6.7 Hz). LRMS: +ve ion 310 [M+Na], −ve ion 286 [M−H].

EXAMPLE 5

[0179] 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-hydroxymethyl-3-methyl-butyl)-amide

[0180] Diastereoisomer A: ¹H-NMR; δ (CD₃OD), 4.07 (1H, m), 3.55 (1H, m),3.45 (2H, m), 3.20 (1H, m), 2.85 (0.5H, m), 2.80 (0.5H, m), 1.60 (3H,m), 1.35 (6H, m) and 0.93 (9H, m). LRMS: +ve ion 289 [M+H], −ve ion 287[M−H].

[0181] Diastereoisomer B: ¹H-NMR; δ (CD₃OD), 4.07 (1H, m), 3.59 (1H, m),3.45 (2H, m), 3.24 (1H, m), 2.70 (1H, m), 1.62 (3H, m), 1.35 (6H, m) and0.93 (9H, m). LRMS: +ve ion 311 [M+Na], 289 [M+H], −ve ion 287 [M−H].

EXAMPLE 6

[0182] 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-hydroxymethyl-2-phenyl-ethyl)-amide

[0183] Diastereoisomer A: ¹H-NMR; δ (CD₃OD), 7.24 (5H, m), 4.15 (1H, m),3.54 (2H, d, J=5.4 Hz), 3.38 (1H, dd, J=7.8, 13.1 Hz), 3.14 (1H, dd,J=4.7, 13.2 Hz), 2.95 (1H, dd, J=7.3, 13.7 Hz), 2.68 (2H, m), 1.58 (2H,m), 1.32 (4H, m), and 0.91 (3H, t, J=6.7 Hz). LRMS: +ve ion 345 [M+Na],323 [M+H], −ve ion 321 [M−H].

[0184] Diastereoisomer B: LRMS: +ve ion 345 [M+Na], 323 [M+H], −ve ion321 [M−H].

EXAMPLE 7

[0185] 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid[2,2-dimethyl-1S-pyridin-2-yI-carbamoyl)-propyl]-amide

[0186] Diastereoisomer A: colourless oil. ¹H-NMR; δ (CD₃OD), 8.34 (1H,m), 8.06 (1H, m), 7.90 (1H, m), 7.33 (1H, m), 4.45 (1H, s), 3.55 (1H,dd, J=8.3, 13.2 Hz), 3.25 (1H, m), 3.05 (1H, m), 1.61 (2H, m), 1.32 (4H,m), 1.11 (9H, s) and 0.85 (3H, m). LRMS: +ve ion 379 [M+H], −ve ion 377[M−H].

[0187] Diastereoisomer B: colourless oil. ¹H-NMR; δ (CD₃OD), 8.33 (1H,m), 8.20 (0.5H, m), 7.93 (1H, m), 7.41 (0.5H, m), 7.28 (1H, m), 4.48(1H, s), 3.52 (1H, dd, J=8.8, 13.1 Hz), 3.23 (1H, dd, J=3.9,13.1 Hz),3.05 (0.5H, m), 2.87 (0.5H, m), 1.62 (2H, m), 1.36 (4H, m), 1.11 (9H, s)and 0.93 (3H, m). LRMS: +ve ion 393 [M+Na], 379 [M+H], −ve ion 377[M−H].

EXAMPLE 8

[0188] 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-dimethylcarbamoyl-2-methyl-propyl) amide

[0189] Diastereoisomer A: colourless oil. LRMS: +ve ion 338 [M+Na], −veion 319 [M−H].

EXAMPLE 9

[0190] 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-dimethylcarbamoyl-2-phenyl-ethyl) amide

[0191] Diastereoisomer A: colourless oil. LRMS: +ve ion 386 [M+Na], −veion 362 [M−H].

[0192] Diastereoisomer B: colourless oil. LRMS: +ve ion 386 [M+Na], −veion 362 [M−H]

EXAMPLE 10

[0193] 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-dimethylcarbamoyl-3-methyl-butyl) amide

[0194] Diastereoisomer A: colourless oil. LRMS: +ve ion 352 [M+Na], −veion 328 [M−H].

EXAMPLE 11

[0195] 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid[3-methyl-1S-pyrrolidine-1-carbonyl)-butyl] amide

[0196] Diastereoisomer A: colourless oil. LRMS: −ve ion 354 [M−H].

EXAMPLE 12

[0197] 1-{2R (orS)-[(Formyl-hydroxy-amino)-methyl]-hexanoyl}-pyrrolidine-2S-carboxylicacid dimethylamide

[0198] Diastereoisomer A: colourless oil. LRMS: +ve ion 336 [M+Na], −veion 312 [M−H].

[0199] Diastereoisomer B: colourless oil. LRMS:+ve ion 336 [M+Na], −veion 312 [M−H].

EXAMPLE 13

[0200] 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-hexanoicacid-(1S-dimethylcarbamoyl-2,2-dimethyl-propyl)-amide

[0201] A synthetic route to the title compound is outlined in Scheme 2and is described in detail below.

[0202] Step A: 2RS-Formyl-heptanoic Acid Ethyl Ester

[0203] Sodium metal (4.38 g, 0.191 mmol) was cut into small pieces andplaced in a two-neck oven-dried round bottom flask under a blanket ofargon. Anhydrous diethyl ether (100 ml) was added and the suspension wasstirred and cooled to 0° C. The flask was fitted with a reflux condenserbefore dropwise addition of ethanol (1.03 ml, 17.3 mmol). A mixture ofethyl formate (15.41 g, 0.208 mmol) and ethyl caproate (25 g, 0.173mmol) was added dropwise via a dropping funnel over a period of about 20minutes. The resulting orange suspension (sodium metal still visible)was allowed to warm to room temperature and stirred overnight. Theresulting thick orange suspension (no sodium metal visible) was cooledto 0° C. and diluted with ice-cold water (100 ml). The mixture wastransferred to a separating funnel and the aqueous phase was removed,washed with diethyl ether, cooled to 0° C. and acidified with 1 Mhydrochloric acid (200 ml). The emulsion was extracted with ethylacetate and the organic layer was separated, washed with brine, driedover anhydrous magnesium sulfate and filtered. The filtrate wasconcentrated under reduced pressure to give a yellow oil containingprimarily the title compound (11.09 g), which was used without furtherpurification in Step B.

[0204] Step B: 2RS-(Benzyloxyimino-methyl)-heptanoic Acid Ethyl Ester

[0205] The crude Claisen product from Step A (11.0 g, 63.9 mmol) wasdissolved in ethanol (100 ml) and water (10 ml) and cooled to 0° C.during the addition of sodium acetate (6.2 g, 76.6 mmol) and O-benzylhydroxylamine hydrochloride (12.23 g, 76.6 mmol). The mixture wasallowed to warm to room temperature and stirred overnight. The resultingsuspension was filtered and the filtrate was concentrated under reducedpressure. The residual yellow paste was partitioned between ethylacetate and water. The organic layer was washed with 1 M hydrochloricacid and brine, dried over anhydrous magnesium sulfate, filtered andevaporated to a yellow oil. The desired product was obtained by flashchromatography (silica gel, gradient elution with 10% to 25% ethylacetate in hexane. Yield 9.19 g (52%). ¹H-NMR: δ (CDCl₃, mixture of syn-and anti-isomers), 7.46 (0.6H, d, J =8.0 Hz), 7.38-7.28 (5H, m), 6.79(0.4H, d, J =7.1 Hz), 5.11 (0.8H, s), 5.08 (1.2H, s), 4.16 (1.2H, q, J=7.0 Hz), 4.13 (0.6H, q, J =7.0 Hz), 3.91 (0.4H, q, J =7.2 Hz), 3.21(0.6H, td, J =8.0 and 6.1 Hz), 1.90-1.48 (2H, m), 1.37-1.20 (7H, m),0.87 (3H, t, J =7.0 Hz).

[0206] Step C: 2RS-(Benzyloxyimino-methyl)-heptanoic acid

[0207] 2RS-(Benzyloxyimino-methyl)-heptanoic acid ethyl ester (7.0 g,25.24 mmol) was dissolved in methanol (125 ml) and the solution wascooled to 0° C. 1 M Sodium hydroxide (26 ml, 26 mmol) was added inportions over 2 minutes to give a pale yellow emulsion. Additionalmethanol was added until a clear solution was obtained. The solution wasallowed to stir for 90 minutes at 0° C. then for 5 hours at roomtemperature whereupon TLC analysis suggested that all of the startingmaterial had been consumed. The solvent was removed under reducedpressure and the residue was partitioned between water and ethylacetate. The aqueous layer was cooled to 0° C. and acidified with 1 Mhydrochloric acid. The emulsion thus formed was extracted twice withethyl acetate. The combined organic extracts were washed with brine,dried over anhydrous magnesium sulfate and filtered. The filtrate wasconcentrated under reduced pressure to provide the title compound as ayellow oil (5.15 g, 82%) which was used without further purification inStep D. ¹H-NMR: δ (CDCl₃, mixture of syn- and anti-isomers), 8.00 (1H,br s), 7.46 (0.6H, d, J =7.9 Hz), 7.36-7.24 (5H, m), 6.80 (0.4H, d, J=7.0 Hz), 5.13 (0.8H, s), 5.09 (1.2H, s), 3.94 (0.4H, q, J =7.1 Hz),3.27 (0.6H, td, J =6.4 and 8.0 Hz), 1.94-1.58 (2H, m), 1.48-1.24 (4H, m)and 0.94-0.84 (3H, m).

[0208] Step D: 2RS-(Benzyloxyimino-methyl)-heptanoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-propyl) Amide

[0209] 2-(Benzyloxyimino-methyl)-heptanoic acid (5.16 g, 20.7 mmol),tert-leucine N,N-dimethylamide (3.60 g, 22.77 mmol) and EDC (4.76 g,24.84 mmol) were stirred together in DMF (75 ml) and cooled to 0° C.HOAt (250 mg, cat.) was added and the bright yellow mixture was allowedto warm to room temperature and stirred overnight. The solvent wasremoved under reduced pressure and the residual oil was partitionedbetween ethyl acetate and 1 M hydrochloric acid. The organic layer waswashed with brine, dried over anhydrous magnesium sulfate, filtered andconcentrated to dryness under reduced pressure. The title compound wasobtained as a colourless oil by flash chromatography (silica gel,gradient elution with 33% to 66% ethyl acetate in hexane). Yield 6.84 g(85%). ¹H-NMR: δ (CDCl₃, mixture of syn- and anti- isomers), 7.45 (0.6H,2d), 7.40-7.26 (5H, m), 6.72 (0.4H, 2d), 6.58 (1H, m), 5.20-4.69 (3H,m), 3.82 (0.4H, m), 3.16-3.10 (3H, m), 3.05 (0.6H, m), 2.99-2.92 (3H,m), 1.90-1.54 (2H, m), 1.39-1.17 (4H, m), 0.97 (2.7H, s), 0.96 (1.8H,s), 0.94 (2.7H, s), 0.92 (1.8H, s) and 0.92-0.82 (3H, m).

[0210] Step E: 2R (or S)-(Benzyloxyamino-methyl)-heptanoic acid(1S-dimethylcarbamoyl -2,2-dimethyl-propyl) Amide

[0211] To a solution of 2RS-(benzyloxyimino-methyl)-heptanoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-propyl) amide (5.0 g, 12.84 mmol) inacetic acid (40 ml) was added sodium cyanoborohydride (2.02 g, 32.0mmol) in one portion. Over the course of 1 hour the reagent dissovedslowly with mild effervesence to give a colourless solution, which wasleft to stir overnight. The solvent was removed by evaporation underreduced pressure and azeotroping with toluene. The remaining oil waspartitioned between diethyl ether and 1 M sodium carbonate (CARE!-somegas evolved). The organic layer was washed with brine (70 ml), washedwith brine, dried over anhydrous magnesium sulfate, filtered andconcentrated to dryness under reduced pressure. The two diastereoisomersof title compound were purified and separated by flash chromatography(silica gel, gradient elution with 50% to 100% ethyl acetate in hexane).

[0212] Diastereoisomer A (faster eluting): colourless oil (2.27 g, 45%).¹H-NMR: δ (CDCl₃), 7.43-7.28 (5H, m), 6.76 (1H, br d, J =9.4 Hz), 5.69(1H, br s), 4.93 (1H, d, J =9.4 Hz), 4.72 (2H, s), 3.15 (3H, s),3.18-3.00 (2H, m), 2.96 (3H, s), 2.49 (1H, m), 1.66-1.49 (2H, m),1.46-1.19 (4H, m), 0.99 (9H, s) and 0.86 (3H, t, J =6.8 Hz).

[0213] Diastereoisomer B (slower eluting): colourless oil (1.44 g, 46%).¹H-NMR: δ (CDCl₃), 7.40-7.27 (5H, m), 6.70 (1H, br d, J =9.0 Hz), 5.99(1H, br s), 4.85 (1H, d, J =9.0 Hz), 4.71 (2H, d, J =1.6 Hz), 3.16 (3H,s), 3.06-2.97 (2H, m), 2.95 (3H, s), 2.57 (1H, m), 1.74-1.21 (6H, m),1.00 (9H, s) and 0.88 (3H, br t, J =6.7 Hz).

[0214] Step F: 2R (or S)-[(Benzyloxy-formyl-amino)-methyl]-heptanoicacid (1S-dimethylcarbamoyl-2,2-dimethyl-propyl) amide

[0215] 2-(Benzyloxyamino-methyl)-heptanoic acid(1S-dimethylcarbamoyl-2,2-dimethylpropyl) amide (diastereoisomer A)(2.02 g, 5.13 mmol) was dissolved in anhydrous THF (50 ml) and placedunder a blanket of argon. N-formyl-benzotriazole (A. R. Katritzky etal., Synthesis 1995, 503) (0.83 g, 5.65 mmol) was added and the mixturewas allowed to stir at room temperature for 4 hours. The solvent wasevaporated under reduced pressure and the residual oil was partitionedbetween dichloromethane and 1 M sodium hydroxide. The organic layer waswashed with more sodium hydroxide and brine, dried over anhydrousmagnesium sulfate, filtered and concentrated to dryness under reducedpressure. The title compound was obtained as a white crystalline solidby flash chromatography (silica gel, elution with 33% ethyl acetate inhexane). Yield 1.60 g (74%). ¹H-NMR: δ (CDCl₃, rotamers), 8.00 (1H, brm), 7.47-7.29 (5H, m), 6.25 (1H, br d, J =9.3 Hz), 5.08-4.74 (2H, br m),4.87 (1H, d, J =9.4 Hz), 3.89-3.52 (2H, br m), 3.13 (3H, s), 2.94 (3H,s), 2.54 (1H, m), 1.67-1.11 (6H, m), 0.95 (9H, s) and 0.85 (3H, br t, J=6.9 Hz).

[0216] 2-(Benzyloxyamino-methyl)-heptanoic acid(1S-dimethylcarbamoyl-2,2-dimethylpropyl) amide (diastereoisomer B) wassimilarly prepared from the the slower eluting diastereoisomer in StepE. Yield 0.38 g (41%). ¹H-NMR: δ (CDCl₃, rotamers), 8.00 (1H, br m),7.47-7.28 (5H, br m), 6.29 (1H, br d, J =9.3 Hz), 5.01-4.63 (2H, br m),4.88 (1H, d, J =9.3 Hz), 3.82-3.51 (1.5H, br m), 3.20-2.78 (6.5H, br m),2.50 (1H, br m), 1.76-1.17 (6H, br m), 0.97 (9H, s) and 0.85 (3H, br t,J =6.7 Hz).

[0217] Step G: 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-hexanoicacid-(1S-dimethyl carbamoyl-2,2-dimethyl-propyl)-amide

[0218] 2-[(Benzyloxy-formyl-amino)-methyl]-heptanoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-propyl) amide (diastereoisomer A)(1.43 g, 3.41 mmol) was dissolved in methanol (50 ml) and placed under ablanket of argon. A suspension of 10% palladium on charcoal (100 mg,cat.) in ethyl acetate (2 ml) was added and the mixture was stirredvigorously while hydrogen gas was bubbled through the solution. After 10minutes the mixture was placed under an atmosphere of hydrogen and leftto stir for 3 hours, whereupon TLC analysis indicated that all of thestarting material had been consumed. The system was purged with argonand the catalyst was removed by filtration. The filtrate wasconcentrated under reduced pressure to provide the title compound as acolourless hygroscopic foam (1.11 g, 99%). ¹H-NMR: δ (CDCl₃, rotamers),8.41 (0.35H, s), 7.83 (0.65H, br s), 6.80 (0.35H, br d, J =8.9 Hz), 6.62(0.65H, br d, J =9.4 Hz), 4.91 (0.65H, br d, J =9.4 Hz), 4.88 (0.35H, brd, J =8.9 Hz), 4.04 (1H, dd, J =14.7 and 7.4 Hz), 3.82 (0.65, dd, J=14.0 and 9.7 Hz), 3.56 (0.35H, dd, J =14.7 and 3.3 Hz), 3.48 (0.65H,dd, J =14.0 and 4.0 Hz), 3.16 (1.05H, s), 3.15 (1.95H, s), 2.98 (1.05H,s), 2.96 (1.95H, s), 2.90-2.74 (0.65H, br m), 2.74-2.61 (0.35H, br m)1.73-1.17 (6H, br m), 0.99 (3.15H, s). 0.95 (5.85H, s) and 0.87 (3H, brt, J =6.7 Hz). ¹³C-NMR; δ (CDCl₃), 174.6, 171.2, 162.2, 157.2, 60.1,54.5, 54.3, 52.3, 48.4, 44.8, 44.3, 35.6, 35.4, 29.6, 29.0, 26.3, 20.8,20.2, 14.0 and 13.7. LRMS: +ve ion 352 (M+Na), −ve ion 328 (M−H).

[0219] 2-[(Formyl-hydroxy-amino)-methyl]-hexanoicacid-(1S-dimethylcarbamoyl-2,2-dimethyl-propyl)-amide (diastereoisomerB) was similarly prepared from diastereoisomer B in Step E ¹H-NMR; δ(CDCl₃, rotamers), 9.37 (0.5H, s), 8.40 (0.5H, s), 7.75 (0.5H, br s),6.62 (0.5H, br s), 6.41 (0.5H, br d, J=7.1 Hz), 4.87 (0.5H, br d, J=6.6Hz), 4.66 (0.5H, br d, J=7.6 Hz), 3.84-3.39 (2H, m), 3.21 (1 .5H, br s),3.14 (1.5H, br s), 2.98 (3H, br s), 2.91-2.54 (1H, m), 1.79-1.23 (6H, m)and 1.08-0.83 (12H, m). ¹³C-NMR; δ (CDCl₃, rotamers), 174.9,173.3, 56.3,54.8, 51.6, 50.3, 45.5, 45.1, 38.6, 38.4, 36.2, 36.0, 35.3, 34.4, 29.5,29.4, 29.3, 29.2, 26.6, 26.5, 22.6, 22.5 and 13.9. LRMS: +ve ion 352[M+Na], −ve ion 328 [M−H].

Method II

[0220] An alternative asymmetric synthetic route to the compound ofExample 13 is outlined in Scheme 3 and is described in detail below.

[0221] Step A: 2-Butyl Acrylic Acid

[0222] To a solution of n-butylmalonic acid (17.2 g, 107 mmol) inethanol (200 ml) was added piperidine (12.76 ml, 129 mmol) and 37% aq.formaldehyde (40.3 ml, 538 mmol). The solution was heated to 80° C.during which time a precipitate appeared and then gradually redissolvedover 1 hour. The reaction mixture was stirred at 80° C. overnight thencooled to room temperature. The solvents were removed under reducedpressure and the residue was dissolved in ethyl acetate (200 ml), washedsuccessively with 1 M hydrochloric acid and brine, dried over anhydrousmagnesium sulfate and filtered. The filtrate was concentrated to givethe title compound as a clear oil (13.37 g, 97%). ¹H-NMR; δ (CDCl₃),6.29 (1H, s), 5.65 (1H, s), 2.34-2.28 (2H, m), 1.54-1.26 (4H, m) and0.94 (3H, t, J=7.1 Hz).

[0223] Step B:4S-Benzyl-3-(2-butyl-acryloyl)-5,5-dimethyl-oxazolidin-2-one

[0224] 2-Butyl acrylic acid (21.5 g, 168 mmol) was dissolved in dry THF(500 ml) and cooled to −78° C. under a blanket of argon. Triethylamine(30 ml, 218 mmol) and pivaloyl chloride (21 ml, 168 mmol) were added atsuch a rate that the temperature remained below −60° C. The mixture wasstirred at −78° C. for 30 minutes, warmed to room temperature for 2hours and finally cooled back to −78° C.

[0225] In a separate flask, 4S-benzyl-5,5-dimethyl-oxazolidin-2-one wasdissoved in dry THF (500 ml) and cooled to -78° C. under a blanket ofargon. n-Butyllithium (2.4 M solution in hexanes, 83 ml, 200 mmol) wasadded slowly and the mixture was stirred for 30 minutes at roomtemperature. The resulting anion was tranferred via a cannula into theoriginal reaction vessel. The mixture was allowed to warm to roomtemperature and was stirred overnight at room temperature. The reactionwas quenched with 1 M potassium hydrogen carbonate (200 ml) and thesolvents were removed under reduced pressure. The residue waspartitioned between ethyl acetate and water. The organic layer waswashed with brine, dried over anhydrous magnesium sulphate, filtered andconcentrated under reduced pressure to give an orange oil. TLC analysisrevealed the presence of unreacted chiral auxiliary in addition to therequired product. A portion of the material (30 g) was dissolved indichloromethane and flushed though a silica pad to give pure titlecompound as a yellow oil (25.3 g). ¹H-NMR;δ (CDCl₃), 7.31-7.19 (5H, m),5.41 (2H,s), 4.51 (1H, dd, J=9.7, 4.2 Hz), 3.32 (1H, dd, J=14.2, 4.2Hz), 2.82 (1H, dd, J=14.2, 9.7 Hz), 2.40-2.34 (2H, m), 1.48-1.32 (4H,m), 1.43 (3H, s), 1.27 (3H, s) and 0.91 (3H, t, J=7.1 Hz). Some chiralauxiliary was recovered by flushing the silica pad with methanol.

[0226] Step C:4S-Benzyl-3-[2-(benzyloxyamino-methyl)-hexanoyl]-5,5-dimethyl-oxazolidin-2-one(p-toluenesulfonic acid salt)

[0227] 4S-Benzyl-3-(2-butyl-acryloyl)-5,5-dimethyl-oxazolidin-2-one(19.8 g, 62.8 mmol) was mixed with O-benzylhydroxylamine (15.4 g, 126mmol) and stirred overnight at room temperature. The mixture wasdissolved in ethyl acetate and the solution was washed with 1 Mhydrochloric acid, 1 M sodium carbonate and brine, dried over anhydrousmagnesium sulfate and filtered. The filtrate was concentrated underreduced pressure to a pale yellow oil (25.3 g) which was shown by NMRand HPLC analysis to contain4S-Benzyl-3-[2-(benzyloxyamino-methyl)-hexanoyl]-5,5-dimethyl-oxazolidin-2-one(ca. 82% d.e.) along with a trace of starting material. The product wascombined with another batch (26.9 g, 76% d.e.) and dissolved in ethylacetate (200 ml). p-Toluenesulfonic acid (22.7 g, 119 mmol) was addedand the mixture was cooled to 0° C. The title compound was obtained as awhite crytalline solid by seeding and scratching. Yield: 25.2 g, (34%,single diastereoisomer). A second crop (14.7 g, 20%, singlediastereoisomer) was also obtained. ¹H-NMR;δ (CDCl₃), 7.89 (2H, d, J=8.2Hz), 7.37-7.12 (1 OH, m), 7.02 (2H, d, J=6.9 Hz), 5.28-5.19 (2H, m),4.55 (1H, m), 4.23 (1H, m), 3.93 (1H, m), 3.58 (1H, m), 2.58 (1H, m),2.35 (3H, s), 1.67-1.51 (2H, m), 1.29-1.16 (4H, m), 1.25 (3H, s), 1.11(3H, s) and 0.80-0.75 (3H, m).

[0228] Step D: 2R-Benzyloxyamino-methyl)-hexanoic Acid

[0229]4S-Benzyl-3-[2R-(benzyloxyamino-methyl)-hexanoyl]-5,5-dimethyl-oxazolidin-2-onep-toluenesulfonic acid salt (25.2 g, 40.2 mmol) was partitioned betweenethyl acetate and 1 M sodium carbonate. The organic phase was dried overanhydrous magnesium sulfate, filtered and evaporated under reducedpressure. The residual oil was dissolved in THF (150 ml) and water (50ml) and cooled to 0° C. and treated with lithium hydroxide (1.86 g, 44.2mmol). The solution was stirred for 30 minutes at 0° C, then overnightat room temperature. The reaction was acidified to pH4 with 1 M citricacid and the solvents were removed. The residue was partitioned betweendichloromethane and 1 M sodium carbonate. The basic aqueous layer wasacidified to pH4 with 1 M citric acid and extracted three times withethyl acetate. The combined organic layers were dried over anhydrousmagnesium sulfate, filtered and concentrated to provide the titlecompound as a colourless oil (7.4 g, 73%). ¹H-NMR;δ (CDCl₃), 8.42 (2H,br s), 7.34-7.25 (5H, m), 4.76-4.66 (2H, m), 3.20-3.01 (2H, m), 2.73(1H, m), 1.70-1.44 (2H, m), 1.34-1.22 (4H, m) and 0.92-0.86 (3H, m).

[0230] Step E: 2R-(Benzyloxyamino-methyl)-hexanoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-1-propyl) Amide

[0231] 2R-Benzyloxyamino-methyl)-hexanoic acid (7.98 g, 31.8 mmol) wasdissolved in DMF (150 ml) and the solution was cooled to 0° C. EDC (6.1g, 31.8 mmol) and HOBt (430 mg, 3.2 mmol) were added and the mixture wasstirred for 15 minutes. tert-Leucine-N,N-dimethylamide (5.53 g, 34 mmol)was added and the reaction was allowed to warm to room temperature andwas stirred overnight. The solvent was removed under reduced pressureand the residue was dissolved in ethyl acetate, washed successively with1 M hydrochloric acid, saturated sodium hydrogen carbonate and brine,dried and filtered. The solvent was removed to leave the title compoundas a yellow oil (8.7 g, 69%) which was used in Step F without furtherpurification. ¹H-NMR; δ (CDCl₃), 7.35-7.28 (5H, m), 6.77 (1H, br d,J=9.2 Hz), 5.69 (1H, br s), 4.93 (1H, d, J=9.4 Hz), 4.72 (2H, s), 3.15(3H, s), 3.10-3.00 (2H, m), 2.95 (3H, s), 2.49 (1H, m), 1.64-1.21 (6H,m), 0.99 (9H, s) and 0.86 (3H, t, J=6.8 Hz).

[0232] Step F: 2R-[(Benzyloxy-formyl-amino)-methyl]-hexanoic acid(1S-dimethyl-carbamoyl-2,2-dimethyl-1-propyl) Amide

[0233] 2R-(Benzyloxyamino-methyl)-hexanoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-1-propyl) amide (7.8 g, 19.9 mmol)was dissolved in dry THF (100 ml) and treated with1-formyl-benzotriazole (3.08 g, 21.0 mmol). The reaction was stirredovernight at room temperature. The solvent was removed under reducedpressure and the residue was dissolved in ethyl acetate, washed with 2 Msodium hydroxide solution and brine. The organic layer was dried overanhydrous magnesium sulfate, filtered and concentrated to dryness underreduced pressure. The product was crystallised from ether-hexane (4.83g, 57% in two crops). ¹H-NMR;δ (CDCl₃, rotamers), 8.12 (0.6H, br s),7.89 (0.4H, br s), 7.37 (5H, s), 6.25 (1H, d, J=9.3 Hz), 4.96 (0.6H, brs), 4.86 (1H, d, J=9.4 Hz), 4.80 (0.4H, br s), 3.74 (2H, br s), 3.13(3H, s), 2.94 (3H, s), 2.53 (1H, m), 1.38-1.21 (6H, m), 0.95 (9H, s) and0.85 (3H, t, J=6.9 Hz). Note: A small sample was crytallised fromether-hexane to provide crystals suitable for X-ray crystallography. Thestereochemistry was proven to be as stated herein.

[0234] Step G: 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-1-propyl) Amide

[0235] 2R-[(Benzyloxy-formyl-amino)-methyl]-hexanoic acid(1S-dimethylcarbamoyl-2,2-dmethyl-1-propyl) amide (4.72 g, 11.3 mmol)was dissolved in ethanol (80 ml) and placed under a blanket of argon. Asuspension of 10% palladium on charcoal (940 mg) in ethyl acetate (2 ml)was added and the mixture was stirred vigorously as hydrogen gas wasbubbled through the system. After 30 minutes the suspension was placedunder a balloon of hydrogen and stirred overnight at room temperature.The flask was purged with argon before removing the catalyst byfiltration. The filtrate was concentrated under reduced pressure toprovide the title compound as a colourless foam which crystallised onstanding (3.65 g, 98%). ¹H-NMR; δ (CDCl₃, rotamers), 9.32 (0.4H, br s),8.41 (0.4H, s), 7.88 (0.6H, br s), 7.27 (0.6H, s), 6.75 (0.4H, br d,J=8.8 Hz), 6.58 (0.6H, br d, J=9.3 Hz), 4.89 (1H, m), 4.04 (0.4H, m),3.82 (0.6H, m), 3.53 (1H, m), 3.16 (1.2H, s), 3.15 (1.8H, s), 2.98(1.2H, s), 2.96 (1.8H, s), 2.79 (0.6, m), 2.65 (0.4H, m), 1.78-1.58 (6H,m), 0.99 (3.6H, s), 0.95 (5.4H, s) and 0.87, 3H, t, J=6.7 Hz). ¹³C-NMR;δ(CDCl₃, rotamers), 175.8, 173.3,172.0, 55.4, 54.9, 52.2, 48.8, 46.3,38.9, 38.8, 36.3, 36.1, 30.3, 30.2, 29.7, 26.9, 23.0 and 14.3. LRMS: +veion 352 [M+Na], −ve ion 328 [M−H].

[0236] The compounds of Examples 14 to 27 were prepared by analogy withExample 13, Method I, substituting the appropriate ester for ethylcaproate in Step A. Where both diastereoisomers were prepared,diastereoisomer A is the faster eluting and usually the more potentagainst PDF in vitro. In some cases only the faster runningdiastereoisomer (Step E) was taken through to the final product.

EXAMPLE 14

[0237] 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-3-cyclopentyl-propionicacid (1S-dimethyl-carbamoyl-2,2-dimethyl-propyl)-Amide

[0238] Diastereoisomer A. Colourless glass. ¹H-NMR; δ (CDCl₃, rotamers),9.33 (0.4H, br s), 8.94 (0.6H, br s), 8.40 (0.4H, s), 7.82 (0.6H, s),6.82 (0.4H, br d, J=8.6 Hz), 6.62 (0.6H, br d, J=9.3 Hz), 4.90 (1H, m),4.06 (0.4H, br dd, J=14.7, 7.3 Hz), 3.81 (0.6H, br dd, J=14.0, 9.7 Hz),3.50 (1H, m), 3.16 (1.2H, s), 3.14 (1.8H, s), 2.97 (1.2H, s), 2.95(1.8H, s), 2.80 (1H, m), 1.87-1.32 (9H, m), 1.16-0.95 (2H, m), 0.99(3.6H, s) and 0.95 (5.4H, s). ¹³C-NMR; δ (CDCl₃, rotamers), 172.9,171.3,55.0, 54.5, 52.0, 48.6, 45.4, 44.2, 38.5, 38.4, 37.9, 37.6, 36.4, 36.3,35.8, 35.6, 35.5, 32.7, 32.6, 26.5, 26.4 and 25.1. LRMS: +ve ion 378[M+Na], −ve ion 354 [M−H].

[0239] Diastereoisomer B. Colourless glass. ¹H-NMR; δ (CDCl₃, rotamers),9.30 (0.6H, br s), 8.41 (0.6H, s), 7.75 (0.4H, s), 6.52 (0.4H, br d,J=8.7 Hz), 6.41 (0.6H, br d, J=7.3 Hz), 4.85 (0.4H, br d, J=9.5 Hz),4.63 (0.6H, br d, J=7.5 Hz), 3.85-3.40 (2H, m), 3.25-2.95 (6H, 3br s),2.78 (1H, 2br m), 1.90-1.40 (8H, m), 1.30 (1H, m), 1.20-1.00 (2H, m) and1.05-0.95 (9H, 2s). ¹³C-NMR; δ (CDCl₃, rotamers), 174.9, 173.3,172.8,56.5, 54.7, 51.5, 50.5, 44.7, 44.6, 38.6, 38.4, 38.0, 37.8, 36.2, 36.0,35.7, 35.5, 35.3, 34.3, 33.0, 32.9, 32.4, 32.3, 30.9, 26.6, 26.5, 25.1,25.0 and 24.9. LRMS: +ve ion 378 [M+Na], −ve ion 354 [M−H].

EXAMPLE 15

[0240] 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-heptanoicacid-(1S-dimethylcarbamoyl-2,2-dimethyl-propyl)-amide

[0241] Diastereoisomer A. Dark orange oil. ¹H-NMR; δ (CDCl₃, rotamers),8.32 (0.33H, s), 7.76 (0.67H, br s), 6.78 (0.33H, br d, J=9.1 Hz), 6.68(0.67H, br d, J=9.1 Hz), 4.87-4.79 (1H, m), 3.96 (0.33H, br dd, J=1 4.6,7.6 Hz), 3.74 (0.67H, br dd, J=1 3.9, 9.7 Hz), 3.51-3.36 (1H, m), 3.09(1H, s), 3.08 (2H, s), 2.90 (1H, s), 2.89 (2H, s), 2.86-2.55 (1H, m),1.53-1.19 (8H, br m), 0.92 (3H, s), 0.88 (6H, s) and 0.79 (3H, m).¹³C-NMR; δ (CDCl₃, rotamers), 174.3, 172.0, 170.5, 170.4, 54.0, 53.5,53.4, 50.8, 49.7, 47.4, 44.9, 43.8, 37.5, 37.4, 34.8, 34.7, 34.6, 30.6,29.2, 29.1, 25.8, 25.5, 21.4 and 12.9. LRMS: +ve ion 344 M+H], −ve ion342 [M−H].

[0242] Diastereoisomer B. Dark orange oil. ¹H-NMR; δ (CDCl₃, rotamers),8.36 (0.5H, s), 7.74 (0.5H, s), 6.69 (0.5H, br s), 6.57 (0.5H, br d,J=7.6 Hz), 4.89 (0.5H, br s), 4.70 (0.5H, d, J=7.8 Hz), 3.76-3.40 (2H,m), 3.21 (1.5H, s), 3.16 (1.5H, s), 2.98 (3H, s), 2.81 (1H, br s),2.72-2.60 (1H, m), 1.67 (2H, br s), 1.42-1.22 (6H, m), 1.02 (4.5H, s),0.99 (4.5H, s), 0.90 (1.5H, s) and 0.87 (1.5H, s). ¹³C-NMR; δ (CDCl₃,rotamers), 175.2,173.8,173.1, 56.5, 55.1, 52.3, 51.1, 50.6, 45.8, 45.5,39.0, 38.9, 36.6, 36.3, 35.6, 34.9, 32.1, 32.0, 30.1, 29.9, 27.4, 27.4,27.0, 26.9, 22.9 and 14.3. LRMS: +ve ion 344 [M+H], −ve ion 342 [M−H].

EXAMPLE 16

[0243] 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-pentanoicacid-(1S-dimethylcarbamoyl-2,2-dimethyl-propyl)-amide

[0244] Diastereoisomer A. White hygroscopic foam. ¹H-NMR; δ (CDCl₃,rotamers), 8.40 (0.33H, s), 7.83 (0.67H, br s), 6.88 (0.33H, br d, J=8.6Hz), 6.69 (0.67H, br d, J=9.2 Hz), 4.90 (1H, t), 4.06 (0.33H, br dd, J=14.5, 7.4 Hz), 3.82 (0.67H, br dd, J=1 3.7, 9.8 Hz), 3.57-3.44 (1H, m),3.16 (1H, s), 3.15 (2H, s), 2.98 (1H, s), 2.96 (2H, s), 2.87-2.63 (1H,m), 1.64-1.26 (4H, br m), 0.98 (3H, s), 0.94 (6H, s) and 0.90 (3H, t,J=7.3 Hz). ¹³C-NMR; δ (CDCl₃, rotamers), 175.8, 173.2,172.0, 55.4, 54.9,52.2, 48.7, 46.2, 45.0, 38.9, 38.9, 36.3, 36.1, 36.1, 32.7, 32.6, 27.0,26.9, 20.9, 20.8 and 14.4. LRMS: +ve ion 338 [M+Na], −ve ion 314 [M−H].

EXAMPLE 17

[0245] 2R (or S)-[Formyl-hydroxy-amino)-methyl]-4-methyl-pentanoic acid-(1S-dimethyl-carbamoyl-2,2-dimethyl-propyl)-amide

[0246] Diastereoisomer A. White hygroscopic solid. ¹H-NMR; δ (CDCl₃,rotamers), 8.41 (0.4H, s), 7.83 (0.6H, s), 6.65 (0.4H, d, J=8.6 Hz),6.55 (0.6H, d, J=9.0 Hz), 4.91-4.83 (1H, m), 4.03-3.95 (0.4H, m),3.84-3.74 (0.6H, m), 3.62-3.43 (1H, m), 3.16 (1H, s), 3.13 (2H, s), 2.98(1H, s), 2.96 (2H, s), 2.89-2.79 (0.6H, m), 2.76-2.71 (0.4H, m),1.69-1.34 (1.8H, m), 1.29-1.20 (1.2H, m), 1.0 (3.6H, s), 0.95 (5.4H, s)and 0.93-0.88 (6H, m). ¹³C-NMR; δ (CDCl₃, rotamers), 175.8, 173.3,172.0, 171.7, 55.5, 55.0, 52.4, 49.1, 44.3, 43.2, 39.5, 39.4, 38.9,38.8, 36.3, 36.1, 27.0, 26.9, 26.3, 26.0, 23.1, 23.0 and 22.8. LRMS: +veion 352 [M+Na], −ve ion 328 [M−H].

EXAMPLE 18

[0247] 3-Cyclohexyl-2R (or S)-[(formyl-hydroxy-amino)-methyl]-propionicacid (1S-dimethyl-carbamoyl-2,2-dimethyl-propyl)-amide

[0248] White solid. ¹H-NMR; δ (CDCl₃, rotamers), 8.38 (0.25H, s), 7.82(0.75H, s), 6.93 (0.25H, d, J=8.9 Hz), 6.74 (0.75H, d, J=8.9 Hz), 4.90(1H, d, J=9.4 Hz), 4.02 (0.25H, dd, J=9.7, 14.1 Hz), 3.78 (0.75H, dd,J=9.7, 14.1 Hz), 3.46 (1H, m), 3.15 (3H, s), 2.96 (3H, s), 2.92 (1H, m),1.65 (6H, m), 1.20 (5H, m), 0.98 (9H, s) and 0.87 (2H, m). ¹³C-NMR; δ(CDCl₃, rotamers), 176.4, 174.2, 172.4, 56.0, 55.6, 53.4, 49.9, 44.0,43.3, 39.6, 39.4, 38.7, 38.5, 36.9, 36.7, 36.6, 34.8, 34.5, 27.5, 27.4and 27.2. LRMS: +ve ion 370 [M+H], 368 [M−H].

EXAMPLE 19

[0249] 2R (or S)-Cyclopentyl-3-(Formyl-hydroxy-amino)-propionicacid-(1S-dimethylcarbamoyl-2,2- dimethyl-propyl)-amide

[0250] Diastereoisomer A. Off-white foam. ¹H-NMR; δ (CD30D, rotamers),8.22 (0.33H, s), 7.79 (0.66H, s), 4.89 (1H, s), 3.87 (1H, m), 3.50 (1H,m), 3.19 (3H, s,), 2.93 (3H, s), 2.82 (0.66H, m), 2.65 (0.33H, m), 1.89(2H, m), 1.56 (5H, m), 1.24 (2H, m) and 0.98 (9H, s). ¹³C-NMR; δ (CD₃OD,rotamers), 176.0, 56.7, 53.2, 51.1, 42.7, 39.2, 36.5, 36.4, 32.0, 27.4,26.4 and 26.2. IR (reflection disc) v_(max) 3318, 2953,1663,1628,1529,1367,1229,1142,1087, 877 cm⁻¹. LRMS: +ve ion 364 [M+Na], −ve ion340

EXAMPLE 20

[0251] 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-octanoic acid(1S-dimethylcarbamoyl-2,2-dimethyl- propyl)-amide

[0252] Diastereoisomer A. ¹H-NMR; δ (CDCl3, rotamers), 8.40 (0.4H, s),7.83 (0.6H, s), 6.88 (0.4H, d, J=8.9 Hz), 6.68 (0.6H, d, J=9.2 Hz), 4.90(1H, m), 4.05 (0.4H, m), 3.81 (0.6H, m), 3.50 (1H, m), 3.16 (1.2H, s),3.15 (1.8H, s), 2.97 (1.2H, s), 2.96 (1.8H, s), 2.86 (0.6H, m), 2.69(0.4H, m), 1.59-1.25 (1OH, m), 1.14-0.95 (9H, m) and 0.89-0.77 (3H, m).¹³C-NMR; δ (CDCl₃, rotamers), 175.2, 172.9, 171.6, 171.4, 54.9, 54.5,54.3, 52.0, 48.4, 46.1, 45.7, 45.1, 44.7, 39.7, 38.5, 38.4, 35.8, 35.6,35.6, 31.7, 31.5, 30.2, 30.1, 29.1, 29.1, 27.0, 26.4, 22.4 and 14.0.LRMS: +ve ion 380 [M+Na], 358 [M+H], −ve ion 356 [M−H].

EXAMPLE 21

[0253] 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-nonanoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-propyl)-amide

[0254] DiastereoisomerA: brown solid. ¹H-NMR; δ (CDCl₃, rotamers), 9.30(0.4H, s), 8.41 (0.6H, s), 7.83 (0.4H, s), 6.66 (0.4H, d, J=8.9 Hz),6.52 (0.6H, d, J=9.7 Hz), 4.92-4.84 (1H, m), 4.06-3.97 (0.4H, m),3.87-3.77 (0.6H, m), 3.63-3.45 (1H, m), 3.16 (1.2H, s), 3.14 (1.8H, s),2.98 (1.2H, s), 2.96 (1.8H, s), 2.86-2.74 (0.6H, m), 2.66-2.63 (0.4H,m), 1.95-1.25 (12H, m), 1.00-0.95 (9H, m), and 0.90-0.84 (3H, m).¹³C-NMR; δ (CDCl₃, rotamers), 175.5,172.8, 171.4,162.2,156.1, 55.1,54.5, 51.3, 50.8, 48.4, 46.3, 44.9, 38.4, 38.4, 35.8, 35.7, 33.9, 31.7,30.3, 30.2, 29.4, 29.0, 27.1, 26.5, 26.5, 24.9, 22.6 and 14.0. LRMS: +veion 394 [M+Na], 372 [M+H], −ve ion 370 [M−H].

EXAMPLE 22

[0255] 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-decanoic acid(1S-dimethylcarbamoyl-2,2-dimethyl- propyl)-amide

[0256] Diastereoisomer A: colourless oil. LRMS: +ve ion 408 [M+Na], 386[M+H], −ve ion 384 [M−H].

EXAMPLE 23

[0257] 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-5-methyl-hexanoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-propyl)-amide

[0258] Diastereoisomer A: colourless oil. ¹H-NMR; δ (CDCl₃, rotamers),9.31 (0.4H, s), 8.40 (0.4H, s), 8.17 (0.6H, s), 6.77 (0.4H, d, J=7.5Hz), 6.60 (0.6H, d, J=8.0 Hz), 4.89 (1H, m), 4.04 (0.4H, m), 3.83 (0.6H,m), 3.52 (1H, m), 3.16 (1.2H, s), 3.15 (1.8H, s), 2.98 (1.2H, s), 2.96(1.8H, s), 2.70 (1H, m), 1.58-1.14 (5H, m), 1.00-0.95 (9H, m) and0.87-0.84 (6H, m). ¹3C-NMR; δ (CDCl₃, rotamers), 172.9,171.5,162.2,156.3,55.1, 54.6, 51.4, 48.5, 46.4, 45.0, 38.5, 38.4, 36.2, 35.9, 35.6,29.7, 28.1, 28.0, 27.9, 26.7, 26.6, 26.5 and 22.4. LRMS: +ve ion 366[M+Na], 344 [M+H], −ve ion 342 [M−H].

EXAMPLE 24

[0259] 2R (or S)-[(Formyl-hydroxy-amino)-methyl] propanoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-propyl)-amide

[0260] Diastereoisomer A: ¹H-NMR; δ (CDCl₃, rotamers), 8.41 (0.55H, s),7.81 (0.45H, s), 6.67 (0.45H, d, J=8.4 Hz), 6.51 (0.45H, d, J=7.2 Hz),4.88 (0.45H, d, J=9.4 Hz), 4.66 (0.55H, d, J=7.7 Hz), 3.76 (1H, m), 3.55(0.55H, dd, J=14.3, 9.8 Hz), 3.44 (0.45H, dd, J=14.2, 5.3 Hz), 3.21(1.65H, s), 3.14 (1.35H, s), 2.99 (1.65H, s), 2.97 (1.35H, s), 2.81 (1H,m), 1.21 (1.65H, d, J=6.7 Hz), 1.19 (1.35H, d, J=6.8 Hz), 1.01 (4.95H,s) and 0.98 (4.05H, s). LRMS: +ve ion 310 [M+Na], −ve ion 286 [M−H].

[0261] Diastereoisomer B: ¹H-NMR; δ (CDCl₃, rotamers), 9.47 (0.4H, brs), 8.41 (0.4H, s), 7.86 (0.6H, s), 6.96 (0.4H, br s), 6.74 (0.6H, d,J=7.3 Hz), 4.91 (1H, m), 3.99 (0.4H, dd, J=14.2, 7.6 Hz), 3.83 (0.6H,dd, J=13.8, 9.0 Hz), 3.50 (1H, m), 3.16 (1.2H, s), 3.15 (1.8H, s), 2.97(3H, s), 2.90 (1H, m), 1.21 (1.2H, d, J=6.8 Hz), 1.15 (1.8H, d, J=6.5Hz), 0.99 (3.6H, s) and 0.95 (5.4H, s). LRMS: +ve ion 310 [M+Na], −veion 286 [M−H].

EXAMPLE 25

[0262] 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-3-methyl butyric acid(1S-dimethylcarbamoyl-2,2-dimethyl- propyl)-amide

[0263] Diastereoisomer A: ¹H-NMR; δ (CDCl₃, rotamers), 9.33 (0.4H, s),8.38 (0.4H, s), 7.81 (0.6H, s), 6.86 (0.4H, br s), 6.58 (0.6H, d, J=8.6Hz), 4.90 (1H, m), 4.06 (0.4H, dd, J=14.7, 7.3 Hz), 3.91 (0.6H, dd,J=13.8, 10.6 Hz), 3.17 (1.2H, s), 3.15 (1.8H, s), 2.98 (1 .2H, s), 2.96(1 .8H, s), 2.62 (0.6H, m), 2.48 (0.4H, m), 1.90 (1H, m), 1.09-0.86 (15H, m). LRMS: +ve ion 338 (M+Na), −ve ion 314 (M−H).

EXAMPLE 26

[0264] 2R (orS)-[(Formyl-hydroxy-amino)-methyl]-3-phenyl-propionylpropionicacid-(1S-dimethylcarbamoyl-2,2-dimethyl-propyl)-amide

[0265] Diastereoisomer A. Colourless glass. ¹H-NMR; δ (CDCl₃, rotamers),9.33 (0.3H, br s), 8.95 (0.7H, br s), 8.43 (0.3H, br s), 7.83 (0.7H, brs), 7.27-7.10 (5H, m), 6.65 (0.3H, br s), 6.45 (0.7H, br d, J=8.2 Hz),4.80-4.70 (1H, m), 4.22-4.10 (0.3H, m), 3.89 (0.7H, dd, J=13.7, 9.6 Hz),3.63-3.47 (1H, m), 3.20-2.69 (3H, m), 3.04 (3H, br s), 2.86 (3H, br s),and 0.87 (9H, br s). ¹³C-NMR; δ (CDCl₃, rotamers), 137.9,137.7,128.8,128.5, 126.6, 54.9, 54.5, 51.3, 48.3, 47.3, 46.6, 38.3,38.2, 36.2, 36.1, 35.8, 35.7, 35.6, 35.5 and 26.4. LRMS: +ve ion 386(M+Na), −ve ion 362 (M−H).

EXAMPLE 27

[0266] 2R (orS)-[(Formyl-hydroxy-amino)-methyl]-3-(4-methoxy-phenyl)-propionicacid-(1S-dimethyl carbamoyl-2,2-dimethyl-propyl)-amide

[0267] Diastereoisomer A: LRMS: +ve ion 416 (M+Na), 394 (M+H), −ve ion392 (M−H).

[0268] The compounds of Examples 28 to 31 were prepared by analogy withExample 13, Method II, substituting the appropriate amino acid amide orbenzyl ester for tert-leucine N,N-dimethylamide in Step E.

EXAMPLE 28

[0269] 2S-{2R-[Formyl-hydroxy-amino)-methyl]-hexanoylamino}-3-phenylPropionic Acid

[0270] White foam. ¹H-NMR; δ (CD₃OD, rotamers), 8.11 (0.35H, s), 7.80(0.65H, s), 7.31-7.16 (5H, m), 4.68 (1H, dd, J=8.7, 5.5 Hz), 3.58 (1H,m), 3.39 (1H, m), 3.19 (1H, m), 2.98 (1H, m), 2.76 (1H, m), 1.55-1.26(6H, m) and 0.90-0.85 (3H, m). ¹³C-NMR; δ (CD₃OD, rotamers), 176.1,175.7, 174.7, 174.5, 138.6, 138.5, 130.3, 129.5, 129.4, 127.7, 55.0,53.3, 49.8, 45.4, 38.4, 38.3, 31.0, 30.8, 30.1, 23.7 and 14.2. IR(reflection disc) Vmax 2932, 2359, 1727, 1660, 1551, 1454, 1381, 1221,882, 701 cm¹. LRMS: +ve ion 359 [M+Na], −ve ion 335 (M−H).

EXAMPLE 29

[0271] 2S-{2R-[Formyl-hydroxy-amino)-methyl]-hexanoylamino}-3,3-dimethylbutyric acid

[0272] White foam ¹H-NMR; δ (CD₃OD, rotamers), 8.25 (0.3H, s), 7.82(0.7H, s), 4.31 (1H, s), 3.83-3.29 (2H, m), 3.10-2.89 (1H, m), 1.54-1.33(6H, m), 1.03 (3H, s), 1.01 (6H, s) and 0.92-0.87 (3H, m). ¹³C-NMR; 6(CD₃OD, rotamers), 174.9,172.9, 61.0, 52.4, 44.2, 44.0, 33.6, 30.1,29.1, 26.2, 22.6 and 13.1. IR(reflection disc) v_(max) 2959,2359,1644,1537, 1371, 1218, 881 and 704 cm¹. LRMS: +ve ion 325 (M+Na),−ve ion 301 (M−H).

EXAMPLE 30

[0273] 2S-[2R-(Formyl-hydroxy-amino)-methyl]-hexanoic acid{1-[(2S-hydroxymethyl-pyrrolidine-1-carbamoyl]-2,2-dimethyl-propyl}-amide

[0274] Colourless oil. ¹H-NMR; δ (CD₃OD, rotamers), 8.26 (0.4H, s), 7.84(0.6H, s), 4.62 (0.4H, d, J=8.2 Hz), 4.39 (0.6H, d, J=8.4 Hz), 4.12 (1H,m), 3.91-3.37 (6H, br m), 2.93 (0.6H, m), 2.78 (0.4H, m), 1.93 (5H, m),1.45 (2H, m), 1.39 (3H, m), 0.97 (3H, br s), 0.95 (3H, br s), and 0.89(3H, t, J=6.7 Hz). ¹³C-NMR; δ (CDCl₃, rotamers), 174.8,172.9, 65.3,65.1, 59.6, 59.5, 55.9, 55.7, 51.9, 47.8, 44.7, 44.0, 31.5, 30.5, 29.3,28.7, 28.1, 27.3,23.8,22.0,21.2,18.7,18.3,17.6,14.7 and 13.3. LRMS: +veion 394 (M+Na), 372 (M+H), −ve ion 370 (M−H).

EXAMPLE 31

[0275] 2S-[2R-(Formyl-hydroxy-amino)-methyl]-hexanoic acid{1-[(2-hydroxy-ethyl)methylcarbamoyl]-2,2-dimethyl-propyl}-amide

[0276] White foam. ¹H-NMR; δ (CD₃OD, rotamers), 8.25 (0.25H, s), 8.03(0.125H, s), 7.82 (0.625H, s), 4.88 (1H, m), 3.83-3.54 (4H, br m), 3.41(2H, m), 3.25 (2H, s), 2.96 (2H, s and m), 1.49 (2H, m), 1.23 (4H, m),1.00 (6H, s), 0.99 (3H, s), and 0.88 (3H, m). ¹³C-NMR; δ (CD₃OD,rotamers), 173.6,164.4, 61.1, 61.0, 56.9,56.5,54.2, 53.9, 52.2, 41.8,38.9, 36.9, 36.3, 35.3, 31.6, 30.8, 27.5, 24.1 and 14.7. LRMS: +ve ion382 [M+Na], −ve ion 358 [M−H].

[0277] The compounds of Examples 32 to 59 were prepared by analogy withExample 7, Method II, substituting the appropriate amine or amino acidamide/benzyl ester for tert-leucine N,N-dimethylamide in Step E. In somecases HOAt was used in Step E and hydrogenolytic deprotection (Step G)was performed under catalytic transfer conditions (cyclohexene,palladium on charcoal in ethanol)

EXAMPLE 32

[0278] 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoicacid-(1R-dimethylcarbamoyl-2,2-dimethyl-propyl)-amide

[0279] Colourless oil. LRMS: +ve ion 330 [M+H], −ve ion 328 [M−H].

EXAMPLE 33

[0280] 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-dimethylcarbamoyl -2S-methylbutyl)-amide

[0281] White foam. LRMS: +ve ion 352 [M+Na], −ve ion 328 [M−H].

EXAMPLE 34

[0282] 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-dimethylcarbamoyl-2-methoxy-2-methyl-propyl)-amide

[0283] From racemic β-hydroxymethylvaline. Diastereoisomer A. Colourlessoil. LRMS: +ve ion 368 [M+Na], 346 [M+H], −ve ion 344 [M−H].Diastereoisomer B. LRMS: +ve ion 368 [M+Na], 346 [M+H], −ve ion 344[M−H].

EXAMPLE 35

[0284] 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-dimethylcarbamoyl-2-hydroxy-2-methyl-propyl)-amide

[0285] Colourless oil. LRMS: +ve ion 354 [M+Na], −ve ion 330 [M−H].

EXAMPLE 36

[0286] 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid[2-(4-chloro-phenyl)-1S-dimethylcarbamoyl-ethyl]-amide

[0287] Colourless oil. LRMS: +ve ion 330 (M+H), −ve ion 328 (M−H).

EXAMPLE 37

[0288] 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid[1S-dimethylcarbamoyl-2-(4-hydroxy-phenyl)-ethyl]-amide

[0289] Colourless oil. LRMS: +ve ion 402 (M+Na), 380 (M+H).

EXAMPLE 38

[0290] 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-dimethylcarbamoyl-2-naphthalen-2-yi-ethyl)-amide

[0291] Colourless oil. LRMS: +ve ion 414 (M+H), −ve ion 412 (M−H).

EXAMPLE 39

[0292] 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(2-cyclohexyl-1S-dimethyl-carbamoyl-ethyl)-amide

[0293] White foam. LRMS: +ve ion 392 (M+Na), 370 (M+H)

Example 40

[0294] 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-dimethylcarbamoyl-phenylmethyl)-amide

[0295] Colourless oil. LRMS: +ve ion 350(M+H), −ve ion 348 (M−H).

EXAMPLE 41

[0296]2-{2R-[(Formyl-hydroxy-amino)-methyl]-hexanoyl}-1,2,3,4-tetrahydro-isoquinoline-3S-carboxylicAcid Dimethylamide

[0297] LRMS: +ve ion 398 (M+Na), 376 (M+H), −ve ion 374 (M−H).

EXAMPLE 42

[0298] 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(4-amino-1S-dimethylcarbamoylbutyl)-amide

[0299] Colourless oil. LRMS: +ve ion 345 (M+H), −ve ion 343 (M−H).

EXAMPLE 43

[0300] 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-dimethylcarbamoyl-2-hydroxy-ethyl)-amide

[0301] Colourless oil. LRMS: +ve ion 326 (M+Na), −ve ion 302 (M−H).N-Hydroxy-N-[2R-(4-methyl-piperazine-1-carbonyl)-hexyl]-formamide

[0302] LRMS: +ve ion 272 [M+H].

EXAMPLE 45

[0303] N-Hydroxy-N-[2R-(morpholine-4-carbonyl)-hexyl]-formamide

[0304] LRMS: +ve ion 281 (M+Na), 259 (M+H), −ve ion 257 (M−H).

EXAMPLE 46

[0305]N-Hydroxy-N-[2R-(2S-hydroxymethyl-pyrrolidine-1-carbonyl)-hexyl]-formamide

[0306] LRMS: −ve ion 271 (M−H).

EXAMPLE 47

[0307] 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-hydroxymethyl-2,2-dimethyl-propyl)-amide

[0308] LRMS: +ve ion 289 (M+H), −ve ion 287 (M−H).

EXAMPLE 48

[0309] 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-methoxymethyl-2,2-dimethylpropyl)-amide

[0310] LRMS: +ve ion 303 (M+H), −ve ion 301 (M−H).

EXAMPLE 49

[0311] 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid[1S-(4-benzyl-piperidine-1-carbonyl)-2,2-dimethyl-propyl]-amide

[0312] LRMS: −ve ion 458 (M−H).

EXAMPLE 50

[0313] 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid[1S-(benzyl-phenethylcarbamoyl)-2,2-dimethyl-propyl]-amide

[0314] LRMS: +ve ion 496 (M+H), −ve ion 494 (M−H).

EXAMPLE 51

[0315] 2S-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid[2,2-dimethyl-1S-(pyrrolidine-1-carbonyl)-propyl]-amide

[0316] LRMS: +ve ion 356 (M+H), −ve ion 354 (M−H).

EXAMPLE 52

[0317] 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid[2,2-dimethyl-lS-(morpholine-4- carbonyl)-propyl]- amide

[0318] LRMS: +ve ion 372 (M+H), −ve ion 370 (M−H).

EXAMPLE 53

[0319] 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid[2,2-dimethyl-1S-(4-methylpiperazine-1-carbonyl)-propyl]-amide

[0320] LRMS: +ve ion 385 (M+H), −ve ion 383 (M−H).

EXAMPLE 54

[0321] 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid[2,2-dimethyl-1S-(4-methyl-piperidine-1-carbonyl)-propyl]-amide

[0322] LRMS: +ve ion 384 (M+H), −ve ion 382 (M−H).

EXAMPLE 55

[0323] 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-cyclohexylcarbamoyl-2,2-dimethyl-propyl)-amide

[0324] LRMS: +ve ion 398 (M+H), −ve ion 396 (M−H).

EXAMPLE 56

[0325] 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid[1S-(4-acetyl-piperidine-1-carbonyl)-2,2-dimethyl-propyl]-amide

[0326] LRMS: +ve ion 412 (M+H), −ve ion 410 (M−H).piperidine-4-carboxylic acid methyl ester

[0327] LRMS: +ve ion 442 (M+H), −ve ion 440 (M−H).

EXAMPLE 58

[0328] 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid[2,2-dimethyl-1S-(octahydroquinoline-1-carbonyl)-propyl]-amide

[0329] LRMS: +ve ion 424 (M+H), −ve ion 422 (M−H).

EXAMPLE 59

[0330] 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid[1S-(3,4-dihydro-2H-quinoline-1-carbonyl)-2,2-dimethyl-propyl]-amide

[0331] LRMS: −ve ion 416 (M−H).

Example 60

[0332]2S-{3-Ethylsulfanymethyl-2R-[(formyl-hydroxy-amino)-methyl]propionylamino}-3,3,N,N-tetramethylbutyramide

[0333] A synthetic route to the title compound is outlined in Scheme 4and is described in detail below.

[0334] Step A: 2-Ethylsulfanylmethyl-acrylic Acid

[0335] A mixture of malonic acid (5.2 g, 50 mmol), paraformaldehyde (3.3g, 110 mmol), dicyclohexylamine, 9.95 ml, 50 mmol) and ethanethiol 4.06ml, 55 mmol) in dioxane (120 ml) was heated at 70° C. for 2 hours. Thesolvents were removed under reduced pressure, the residue wasredissolved in ethyl acetate and the solution was extracted withsaturated aqueous sodium hydrogen carbonate (4×20 ml). The combinedaqueous layers were washed with ethyl acetate (20 ml) then acidifiedwith 1 M hydrochloric acid. The resulting suspension was extracted intodichloromethane and the solution was dried over anhydrous magnesiumsulfate, filtered and evaporated to provide the title compound as awhite solid (3.76 g, 52%). ¹ H-NMR; δ (CDCl₃), 9.89 (1H, br s), 6.35(1H, s), 5.77 (1H, s), 3.39 (2H, s), 2.49 (2H, dd, J=7.4,14.5 Hz) and1.25 (3H, t, J=5.2 Hz).

[0336] Step B:4S-Benzyl-3-(2-ethylsulfanylmethyl-acryloyl)-5,5-dimethyl-oxazolidin-2-one

[0337] 2-Ethylsulfanylmethyl-acrylic acid (3.76 g, 25.8 mmol) wasdissolved in dry THF (75 ml) and cooled to -78° C. under a blanket ofargon. Triethylamine (4.6 ml, 33.5 mmol) and pivaloyl chloride (3.17 ml,25.8 mmol) were added at such a rate that the temperature remained below-60° C. The mixture was stirred at −78° C. for 30 minutes, warmed toroom temperature for 2 hours and finally cooled back to −78° C.

[0338] In a separate flask, 4S-benzyl-5,5-dimethyl-oxazolidin-2-one wasdissoved in dry THF (75 ml) and cooled to -78° C. under a blanket ofargon. n-Butyllithium (2.4M solution in hexanes, 12.9 ml, 30.9 mmol) wasadded slowly and the mixture was stirred for 30 minutes at roomtemperature. The resulting anion was tranferred via a cannula into theoriginal reaction vessel. The mixture was allowed to warm to roomtemperature and stirred overnight at room temperature. The reaction wasquenched with saturated sodium hydrogen carbonate (20 ml) and thesolvents were removed under reduced pressure. The residue waspartitioned between ethyl acetate and water. The organic layer waswashed successively with saturated sodium hydrogen carbonate, 1 Mhydrochloric acid and brine, dried over anhydrous magnesium sulfate,filtered and concentrated under reduced pressure. The residue waspurified by flash chromatography (silica gel, 20% ethyl acetate inhexane) to provide the title compound as a yellow oil (6.5 g, 76%).¹H-NMR; δ (CDCl₃), 7.29 (5H, m), 5.58 (1H, s), 5.49 (1H, s), 4.54 (1H,dd, J=3.9, 9.7 Hz), 3.52 (2H, dd, J=15.8, 3.1 Hz), 3.38 (1H, dd, J=3.9,14.5 Hz), 2.84 (1H, dd, J=4.6,14.3 Hz), 2.52 (2H, dd, J=7.2,14.6 Hz),1.42 (3H, s), 1.29 (3H, s) and 1.22 (3H, t, J=7.5 Hz). LRMS: +ve ion 356(M+Na), 334 (M+H).

[0339] Step C:4S-Benzyl-3-[(2R-tert-butoxyamino-methyl)-3-ethylsulfanylmethyl-propionyl]-5,5-dimethyl-oxazolidin-2-one

[0340]4S-Benzyl-3-(2-ethylsulfanylmethyl-acryloyl)-5,5-dimethyl-oxazolidin-2-one(2.1 g,6.3 mmol) was dissolved in ethanol (10 ml) andO-tert-butyl-hydroxylamine hydrochloride (0.95 g, 7.56 mmol) was added,followed by triethylamine (1.1 ml, 7.87 mmol). The mixture was stirredat 30° C. overnight. The solvents were removed under reduced pressureand the residue was dissolved in ethyl acetate. The organic solution waswashed succesively with 1 M hydrochloric acid, saturated sodium hydrogencarbonate and brine, dried over anhydrous magnesium sulphate andfiltered. The filtrate was concentrated under reduced pressure toprovide the title compound as a pale yellow oil (2.42 g,91%; singlediastereoisomer by HPLC). ¹H-NMR; δ (CDCl₃),7.30 (5H, m),5.09 (1H, brs),4.54 (1H, dd, J=3.5, 9.9 Hz), 4.33 (1H, m), 3.19 (2H, m), 3.08 (1H, dd,J=5.4, 11.8 Hz), 2.80 (3H, m), 2.56 (2H, dd, J=7.4, 14.7 Hz), 1.41 (3H,s), 1.36 (3H, s), 1.23 (3H, t, J=7.3 Hz) and 1.13 (9H, s). LRMS: +ve ion423 (M+H).

[0341] Step D:(2R-tert-butoxyamino-methyl)-3-ethylsulfanylmethyl-propionic Acid

[0342] A solution of4S-Benzyl-3-[(2R-tert-butoxyamino-methyl)-3-ethylsulfanylmethyl-propionyl]-5,5-dimethyl-oxazolidin-2-onein (2.42 g, 5.72 mmol) THF (40 ml) was cooled to 0° C. and a solution oflithium hydroxide (288 mg, 6.86 mmol) in water (10 ml) was added. Themixture was allowed to warm to room temperature then stirred for 5hours. The solvent was removed under reduced pressure and the residuewas partitioned between water and ethyl acetate. The aqueous layer wasremoved and the ethyl acetate layer was washed successively with waterand saturated sodium hydrogen carbonate. The combined aqueous layerswere washed with ethyl acetate (20 ml) before acidifying with 1 Mhydrochloric acid. The resulting emulsion was extracted withdichloromethane (3×20 ml) and the combined organic layers were driedover anhydrous magnesium sulfate, filtered and evaporated to provide thetitle compound as a colourless oil (0.68 g, 50%). ¹H-NMR; δ (CDCl₃),8.03 (2H, brs), 3.21 (2H, d, J=6.1 Hz), 2.89 (2H, m), 2.75 (1H, m), 2.57(2H, dd, J=7.4, 14.8 Hz), 1.26 (3H, t, J=7.4 Hz) and 1.18 (9H, s). LRMS:+ve ion 236 [M+H], −ve ion 234 M−H].

[0343] Step E: A solution of2S-[2R-(tert-butoxy-amino-methyl)-3-ethylsulfanylmethyl-propionylamino}-3,3,N,N-tetramethylbutyramide

[0344] 2R-tert-butoxyamino-methyl)-3-ethylsulfanylmethyl-propionic acid(340 mg, 1.44 mol) was dissolved in DMF (10 ml) andtert-leucine-N,N-dimethylamide (272 mg, 1.73 mmol), HOAt (19.6 mg, 0.14mmol) and EDC (331 mg, 1.73 mmol) were added. The reaction was stirredovernight at room temperature. The solvent was removed under reducedpressure and the residue was dissolved in dichloromethane. The organicsolution was washed successively with 1 M hydrochloric acid, 1 M sodiumcarbonate and brine, dired over anhydrous magnesium sulfate andfiltered. The filtrate was concentrated under reduced pressure toprovide the required product as a colourless oil (440 mg, 82%). ¹H-NMR;δ (CDCl₃), 6.87 (1H, d, J=9.0 Hz), 5.11 (1H, br s), 4.93 (1H, d, J=9.3Hz), 3.15 (3H, s), 3.11 (1H, m), 2.95 (3H, s), 2.79 (3H, m), 2.54 (3H,s), 1.22 (3H, t, J=7.6 Hz), 1.18 (9H, s) and 1.01 (9H, s). LRMS: +ve ion398 [M+Na], 376 [M+1].

[0345] Step F:2S-{2R-[(tert-Butoxy-formyl-amino)-methyl]-3-ethylsulfanylmethyl-propionylamino}-3,3, N, N-tetramethylbutyramide

[0346] A solution of2S-[2R-(tert-butoxy-amino-methyl)-3-ethylsulfanylmethyl-propionylamino}-3,3,N,N-tetramethylbutyramide (220 mg, 0.58 mmol) indichlormethane (5 ml) was cooled to 0° C. and treated with formic aceticanhydride (0.1 ml). The reaction was stirred at room temperature for 4hours, then the solvent was evaporated under reduced pressure. Theresidue was purified by flash chromatography (silica gel, 50% ethylacetate in hexane as eluent) to provide the title compound as acolourless oil (120 mg, 52%). ¹ H-NMR; δ (CDCl₃, rotamers), 8.31 (1H, brs), 6.56 (1H, d, J=9.1 Hz), 4.94 (0.33H, d, J=9.4 Hz), 4.88 (0.67H, d,J=9.2 Hz), 4.08 (0.67H, br m), 3.83 (1.34H, br m), 3.13 (3H, s), 2.95(3H, s), 2.80 (2H, m), 2.61 (1H, dd, J=6.8, 14.0 Hz), 2.49 (2H, dd,J=7.4, 14.7 Hz), 1.29 (9H, s), 1.25 (3H, t, J=7.2 Hz) and 0.99 (9H, s).LRMS: +ve ion 426 [M+Na], 404 [M+H].

[0347] Step G:2S-{3-Ethylsulfanymethyl-2R-[(formyl-hydroxy-amino)-methyl]propionylamino}-3,3,N,N-tetramethylbutyramide

[0348] A solution of2S-{2R-[(tert-butoxy-formyl-amino)-methyl]-3-ethylsulfanylmethyl-propionylamino}-3,3,N,N-tetramethylbutyramide (120 mg, 0.3 mmol) indeuterochloroform (1 ml) was treated with TFA (4 ml) and allowed tostand at 4° C. overnight. The solvents were removed under reducedpressure and residual TFA was removed by azeotroping with toluene. Theresidue was purified by preparative HPLC to provide the title compoundas a colourless oil (40 mg, 38%; 7:2 mixture of diastereomers by HPLC).¹H-NMR; δ (CDCl₃, rotamers), 8.40 (0.33H, s), 7.87 (0.67H, s), 7.24(0.33H, d, J=9.3 Hz), 6.98 (0.67H, d, J=9.3 Hz), 4.91 (0.67H, d, J=9.3Hz), 4.90 (0.33H, d, J=9.3 Hz), 4.07 (0.33H, dd, J=7.5,14.5 Hz), 3.86(0.67H, dd, J=8.8,14.2 Hz), 3.75 (0.67H, m), 3.68 (0.33H, m), 3.16 (1H,s), 3.15 (2H, s), 3.05 (1H, m), 2.96 (3H, s), 2.77 (1H, m), 2.66 (1H,m), 2.52 (2H, dd, J=7.4,14.8 Hz), 1.22 (3H, t, J=7.3 Hz), 0.99 (3H, s)and 0.96 (6H, s). ¹³C-NMR; 6 (CDCl₃, rotamers), 173.3,171.6,171.2, 55.2,54.8, 51.1, 48.5, 45.2, 44.4, 38.5, 38.4,35.9,35.8,35.7,31.7,31.4,26.7,26.6,26.5 and 14.6. LRMS: +ve ion 370[M+Na],348 [M+H], −ve ion 346 [M−H].

[0349] The compound of Example 61 was prepared similarly usingpiperidine in place of ethanethiol in Step A.

EXAMPLE 61

[0350]2-{2-[(Formyl-hydroxy-amino)-methyl]-3-piperidin-1-yl-propionylamino}-3,3,N,N-tetramethyl-butyramide

[0351] White solid (4:1 mixture of diastereoisomers by HPLC). ¹H-NMR; δ(CDCl₃, rotamers), 8.29 (1H, s), 7.95 (1H, br s), 4.87 (1H, d, J=9.1Hz), 4.02 (1H, dd, J=5.0,14.6 Hz), 3.56 (1H, dd, J=8.2, 14.6 Hz), 3.14(3H, s), 2.96 (3H, s), 2.89 (1H, m), 2.69 (1H, m), 2.52 (5H, m), 1.65(4H, m), 1.49 (2H, m) and 0.99 (9H, s). ¹³C-NMR; δ (CDCl₃),172.2,171.3,60.4, 55.0, 54.9, 48.6, 42.4, 38.8, 36.2, 36.1, 27.0,25.6 and 24.3.LRMS: +ve ion 371 [M+H], −ve ion 369 [M−H].

[0352] The compounds of Examples 62 to 65 were prepared by analogy withExample 7, Method II, substituting O-tert-butylhydroxylamine forO-benzylhydroxylamine in Step B and the appropriate amine or amino acidamide/benzyl ester for tert-leucine N,N-dimethylamide in Step E. Finaldeprotection was performed by acidolysis with TFA (see Example 60,above).

EXAMPLE 62

[0353] 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1R-dimethylcarbamoyl-2-methyl-2-methylsulfanyl-propyl)-amide

[0354] Colourless oil. ¹H-NMR; δ (CDCl₃, rotamers), 8.4 (0.5H, s), 7.85(0.5H, s), 7.11 (0.5H, d, J=9.1 Hz), 6.93 (0.5H, d, J=9.1 Hz), 5.15 (1H,d, J=9.4 Hz), 3.90 (0.5H, m), 3.73 (0.5H, m), 3.64 (0.5H, d, J=1 4.3Hz), 3.48 (0.5H, dd, J=1 4.0,3.9 Hz), 3.22 (3H, s), 2.97 (3H, s), 2.83(0.5H, m), 2.70 (0.5H, m), 2.07 (1 .5H, s), 2.04 (1 .5H, s), 1.58 (1H,m), 1.36 (4H, m), 1.32 (3H, s), 1.28 (3H, s) and 0.86 (3H, t, J=6.6 Hz).¹³C-NMR; δ (CDCl₃, rotamers), 175.4,173.5,170.8, 63.6,53.2,53.1, 52.5,49.5,47.5,46.1, 44.9,41.6, 37.5,36.5,36.4, 35.4, 30.2, 29.8, 28.0, 14.3,12.0 and 11.9. LRMS: +ve ion 362 [M+H], −ve ion 360 [M−H].

EXAMPLE 63

[0355] 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(2-benzylsulfanyl-1R-dimethylcarbamoyl-2-methyl-propyl)-amide

[0356] White foam. ¹H-NMR; δ (CDCl₃, rotamers), 8.37 (0.33H, s), 7.81(0.66H, s), 7.31 (5H, m), 7.06 (0.33H, d, J=8.8 Hz), 6.89 (0.66H, d,J=9.3 Hz), 5.20 (1H, d, J=9.3 Hz), 3.94 (0.33H, dd, J=8.3, 14.6 Hz),3.78 (2.66H, m), 3.61 (0.33H, dd, J=3.5, 14.4 Hz), 3.42 (0.66H, dd,J=5.1, 14.9 Hz), 3.21 (3H, s), 3.03 (3H, s), 2.82 (0.66H, m), 2.69(0.33H, m), 1.61 (1H, m), 1.42 (1H, m), 1.37 (3H, s), 1.32 (3H, s), 1.26(4H, m) and 0.86 (3H, t, J=6.6 Hz). ¹³C-NMR; δ (CDCl₃, rotamers), 175.3,173.5,171.0,138.1, 137.4,129.5,129.3, 129.1,129.0,128.9,127.6,127.4,55.9,53.7, 52.5, 51.2, 49.6, 49.5, 46.1, 44.9, 39.0, 38.6,36.6, 36.4, 33.9, 33.7, 30.3, 30.1, 29.7, 26.7, 26.1, 25.7,25.5,24.2,22.9 and 14.3. LRMS: +ve ion 460 [M+Na], 438 [M+H], −ve ion 436 [M−H].

EXAMPLE 64

[0357] 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid[2-benzylsulfanyl-2-methyl-1R-(morpholine-4-carbonyl)-propyl]-amide

[0358] White foam. ¹H-NMR; δ (CDCl₃, rotamers), 8.44 (0.5H, s), 8.37(0.5H, s), 7.30 (5H, m), 6.88 (0.5H, d, J=8.3 Hz), 6.78 (0.5H, d, J=9.2Hz), 5.12 (1H, d, J=9.5 Hz), 3.91 (1H, dd, J=8.2, 14.6 Hz), 3.78 (1H,m), 3.45 (1H, dd, J=4.5, 14.2 Hz), 2.80 (0.5H, m), 2.64 (0.5H, m), 1.61(1H, m), 1.41 (1H, m), 1.36 (3H, s), 1.33 (3H, s), 1.29 (4H, m) and 0.87(3H, t, J=6.8 Hz). ¹³-NMR; δ (CDCl₃, rotamers), 175.5, 173.4, 169.4,137.8, 129.5, 129.3, 129.1, 129.0, 127.8, 127.5, 67.1, 67.0, 53.3, 53.2,51.99, 49.6, 49.5, 49.2, 47.9, 46.5, 45.0, 43.2, 43.0, 34.0, 30.3, 30.2,29.7, 26.8, 26.5, 25.9, 25.8, 22.9 and 14.3. LRMS: +ve ion 502 [M+Na],480 [M+H], −ve ion 478 [M−H].

EXAMPLE 65

[0359] 2-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid[2-benzylsulfanyl-2-methyl-1R (orS)-(4-methyl-piperidine-1-carbonyl)-propyl]-amide

[0360] Diastereoisomer A. White solid. LRMS: +ve ion 514 [M+Na], 492[M+H], −ve ion 490 [M−H].

[0361] Diastereoisomer B. Colourless gum. LRMS: +ve ion 514 [M+Na], 492[M+H], −ve ion 490 [M−H].

[0362] The compounds of Examples 66 to 68 were prepared by analogy withExample 7, Method II, substituting the appropriate malonic acic forbutylmalonic acid in Step A. O-tert-butylhydroxylamine forO-benzylhydroxylamine in Step C. Stereoselectivity in the Michaeladdition was variable. Final deprotection was performed by acidolysiswith TFA (see Example 60, above).

EXAMPLE 66

[0363] 2R-[(Formyl-hydroxy-amino)-methyl]-pent-4-enoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-propyl)-amide

[0364] Single diastereoisomer. ¹H-NMR; δ (CDCl₃, rotamers), 8.40 (0.25H,s), 7.84 (0.75H, s), 7.05 (0.35H, d, J=9.0Hz), 6.74 (0.65H, d, J=9.3Hz), 5.70 (1H, m), 5.03-5.24 (2H, m), 4.88 (1H, dd, J=9.4, 6.7 Hz), 3.98(0.5H, m), 3.81 (0.5H, m), 3.55 (1H, m), 3.14 (3H, s), 2.97(1.3H, s),2.96 (1.7H, s), 2.75-2.92(1H, m), 2.16-2.50 (2H, m), 0.98 (4.5H, s) and0.94 (4.5H, s). LRMS: +ve ion 336 [M+Na], −ve ion 312 [M−H].

EXAMPLE 67

[0365] 2R-[(Formyl-hydroxy-amino)-methyl]-hex-5-enoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-propyl)-amide

[0366] Diastereoisomer A: colourless oil. ¹H-NMR; δ (CDCL₃, rotamers),8.42 (0.45H, s), 7.84 (0.55H, s), 6.78 (0.45H, d, J=8.4 Hz), 6.60(0.55H, d, J=9.3 Hz), 5.74 (1H, m), 5.03 (2H, m), 4.88 (1H, m), 4.14(0.4H, m), 3.81 (0.6H, m), 3.55 (1H, m), 3.16 (1H, s), 3.15 (2H, s),2.98 (1H, s), 2.97 (2H, s), 2.85 (0.7H, m), 2.68 (0.3H, m), 2.07 (2H,m), 1.73 (1 .6H, m), 1.50 (0.4H, m), 0.99 (4H, s) and 0.95 (5H, s).LRMS: +ve ion 350 [M+Na], −ve ion 326 [M−H].

[0367] Diastereoisomer B: colourless oil. ¹H-NMR; δ (CDCL₃, rotamers),8.41 (0.5H, s), 7.75 (0.5H, s), 6.58 (0.5H, d, J=9.1 Hz), 6.36 (0.5H, d,J=9.1 Hz), 5.75 (1H, m), 5.01 (2H, m), 4.86 (0.5 H, d, J=9.5 Hz), 4.64(0.5 H, d, J=7.5 Hz), 3.42-3.82 (2H, m), 3.22 (1 .5H, s), 3.07 (1 .5H,s), 2.99 (3H, s), 2.87 (0.5H, m), 2.66 (0.5H, m), 2.13 (2H, m), 1.81(1H, m), 1.49 (1H, m), 1.02 (4.5H, s) and 1.00 (4.5H, s). LRMS: +ve ion350 [M+Na], −ve ion 326 [M−H].

EXAMPLE 68

[0368] 2R-[(Formyl-hydroxy-amino)-methyl]-hex-4-ynoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-propyl)-amide

[0369] Diastereoisomer A: colourless oil. ¹H-NMR; δ (CDCl₃, rotamers),8.39 (0.4H, s), 7.87 (0.6H, s), 7.20 (0.4H, d, J=8.4 Hz), 6.94 (0.6H, d,J=9.3 Hz), 4.90 (1H, m), 3.66-4.14 (2H, m), 3.16 (2H, s), 3.14 (2H, s),2.96 (3H, s), 2.88 (1H, m), 2.41 (2H, m), 1.77 (3H, m), 1.00 (3.5H, s)and 0.96 (5.5H, s). LRMS: +ve ion 348 [M+Na], −ve ion 324 [M−H].

[0370] Diastereoisomer B: Colourless oil. ¹H-NMR; δ (CDCl₃, rotamers),8.37 (0.5H, s), 7.81 (0.5H, s), 6.87 (1H, m), 4.91 (0.5H, d, J=9.4 Hz),4.79 (0.5H, d, J=8.2 Hz), 3.76 (1.5H, m), 3.63 (0.5H, m), 3.19 (1.5H,s), 3.14 (1.5H, s), 2.98 (3H, s), 2.85 (1H, s), 2.41 (2H, m), 1.77 (3H,m), 1.03 (4.5H, s) and 1.01 (4.5H, s). LRMS: +ve ion 348 [M+Na], −ve ion324 [M−H].

EXAMPLE 69

[0371] 2R-[1R (or S)-(Formyl-hydroxy-amino)-ethyl]-hexanoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-propyl)-amide

[0372] The title compound was prepared according to the route outlinedin Scheme 5 and as described in detail below:

[0373] Step A: 4-Benzyl-3-hexanoyl-oxazolidin-2-one

[0374] 4S-Benzyl-oxazolidin-2-one (14.5 g, 81.7 mmol) was dissolved indry THF (75 ml) under an argon atmosphere. The solution was cooled in anice bath before slow addition of n-butyllithium (1.6 M in hexanes, 56ml, 89.2 mmol). The lithium salt crystallised from the solution as asolid mass and was allowed to warm to room temperature overnight. Theresulting orange suspension was cooled again in an ice bath during theaddition of a cold solution of hexanoyl chloride (10.4 ml, 74.3 mmol) indry THF (50 ml). The mixture was left to warm to room temperature andwas then stirred for 3 hours. The reaction was quenched with 1 M sodiumcarbonate solution (5 ml) and the solvent was removed under reducedpressure. The residue was partitioned between 1 M sodium carbonate (100ml) and ethyl acetate (150 ml). The organic layer was removed and theaqueous layer was extracted with more ethyl acetate. The combinedorganic layers were washed successively with water, 1 M sodium carbonateand brine, dried over anhydrous magnesium sulphate and filtered. Thefiltrate was concentrated to leave an orange oil. Purification by flashchromatography afforded the title compound as a yellow oil (10.21g,50%). ¹H-NMR; δ (CDCl₃), 7.38-7.24 (3H, m),7.24-7.16 (2H, m),4.68(1H,m),4.24-4.12 (2H, m), 3.30(1H, dd, J=13.4,3.2 Hz), 3.02-2.86 (2H, m),2.77(1H, dd, J=13.4,9.6 Hz), 1.77-1.63 (2H, m), 1.44-1.30 (4H, m) and0.92 (3H, br t, J=6.9 Hz).

[0375] Step B:1-(4S-Benzyl-2-oxo-oxazolidin-3-yl)-2R-butyl-butane-1,3-dione

[0376] 4-Benzyl-3-hexanoyl-oxazolidin-2-one (10.2 g, 37.1 mmol) wasdissolved in THF (150 ml) under an argon atmosphere and cooled to −78°C. Lithium hexamethyldisilazide (1 M in THF, 41 ml, 41 mmol) was addedvia a cannula over a few minutes and the resulting green solution wasstirred at −78° C. for 2 hours. Acetyl chloride (3.3 ml, 46.3 mmol) wasadded slowly and the reaction mixture was stirred for 3.5 hours. Asolution of citric acid (3.0 g, 14 mmol) in water (15 ml) was addedquickly to quench the reaction. The solvent was removed under reducedpressure and the residue was partitioned between ethyl acetate andwater, washed with brine, dried over anhydrous magnesium sulphate andfiltered. The filtrate was concentrated to provide the title compound asa yellow oil (12.11 g, contains residual solvent) which was used withoutfurther purification in Step C. ¹H-NMR; δ (CDCl₃), 7.37-7.21 (5H, m),4.68 (1H, m), 4.53 (1H, dd, J=9.6, 3.7 Hz), 4.23-4.13 (2H, m), 3.43 (1H,dd, J=13.5, 3.3 Hz), 2.75 (1H, dd, J=13.5, 9.9 Hz), 2.33 (3H, s), 2.03(1H, m), 1.77 (1H, m), 1.46-1.26 (4H, m) and 0.98-0.86 (3H, m).

[0377] Step C:1-(4S-Benzyl-2-oxo-oxazolidin-3-yl)-2R-butyl-butane-1,3-dione3-(O-benzyl-oxime)

[0378] To a solution of1-(4S-benzyl-2-oxo-oxazolidin-3-yl)-2R-butyl-butane-1,3-dione (12.11 g,38.15 mmol) in water (10 ml) and ethanol (90 ml) was added sodiumacetate (3.75 g, 45.78 mmol) and O-benzyl hydroxylamine hydrochloride(7.31 g, 45.78 mmol). The resulting suspension was left to stir at roomtemperature overnight. The product (7.3 g, 45%, single oxime isomer)crystallised directly from the reaction and was filtered, washed withaqueous ethanol (1:1) and dried under vacuum. Further material (5.31 g,33%, mixture of oxime isomers) was obtained as a yellow oil from themother liquors by acid-base extraction followed by columnchromatography. ¹H-NMR; δ (CDCl₃, major oxime isomer), 7.34-7.20 (8H,m), 7.12-7.07 (2H, m), 5.14-5.02 (2H, m), 4.53 (1H, m), 4.13(1H, dd,J=9.4, 4.0 Hz), 4.04(1H, br t, J=8.4 Hz), 3.91 (1H, dd, J=9.0, 2.7 Hz),3.16 (1H, dd, J=1 3.4,2.9 Hz), 2.09 (3H, s), 1.97 (1H, m), 1.75 (1H, dd,J=1 3.4,10.8 Hz), 1.67 (1H, m), 1.45-1.22 (4H, m) and 0.91 (3H, br t,J=6.9 Hz).

[0379] Step D: 4S-Benzyl-3-[2R-(1R (orS)-benzyloxyamino-ethyl)-hexanoyl]-oxazolidin-2-one

[0380] The mixture of oximes form Step C (5.31 g, 12.5 mmol) wasdissolved in acetic acid (30 ml) and cooled in an ice-water bath beforeaddition of sodium cyanoborohydride (0.8 g, 12.5 mmol) in one portion.Effervescence subsided after a few minutes and a a further portion ofborohydride (0.8 g) was added. The reaction was allowed to warm to roomtemperature and stirred overnight. The acetic acid was removed underreduced pressure and the residue was azeotroped with toluene. Theresulting oil was dissolved in ethyl acetate, washed with water, 1 Msodium carbonate and brine, dried over anhydrous magnesium sulphate andfiltered. The filtrate was evaporated to leave a pale yellow oil whichwas purified by flash chromatography (silica gel, 10% to 25% ethylacetate in hexane as eluant). Yield 3.43 g, 64%). ¹H-NMR; δ (CDCl₃,mixture of α-diastereoisomers), 7.36-7.17 (10H, m), 5.80 (0.45H, brs),5.55 (0.55H, brd, J=8.9 Hz), 4.72-4.59 (3H, m), 4.20-4.05 (2H, m), 3.97(0.45H, m), 3.82 (0.55H, m), 3.47-3.22 (2H, m), 2.45 (1H, m), 1.90-1.48(2H, m), 1.40-1.14 (7H, m) and 0.95-0.84 (3H, m).

[0381] Step E: N-[2R-(4S-Benzyl-2-oxo-oxazolidine-3-carbonyl)-1R (orS)-methyl-hexyl]-N-benzyloxy-formamide

[0382] 4S-Benzyl-3-[2R-(1R (orS)-benzyloxyamino-ethyl)-hexanoyl]-oxazolidin-2-one (3.08 g, 7.3 mmol)was dissolved in dry THF and treated with N-formylbenzotriazole (1.60 g,10.9 mmol). The reaction was stirred for 4 hours at room temperature.The solvent was removed under reduced pressure and the remaining oil waspartitioned between dichloromethane (40 ml) and 1 M sodium hydroxidesolution (30 ml). The organic layer was removed, washed with more sodiumhydroxide then brine, dried over anhydrous magnesium sulphate, filteredand evaporated. Purification by flash chromatography (silica gel, 20% to50% ethyl acetate in hexane) gave the title compound as a pale yellowsolid (2.50 g, 76%. ¹H-NMR; δ (CDCl₃, mixture of a-diastereoisomers androtamers), 8.22 (1H, br m), 7.54-7.13 (10H, m), 5.22-3.92 (7H, br m),3.30 (1H, m), 2.48 (1H, br m), 1.85-1.13 (9H, br m) and 0.93-0.83 (3H,m).

[0383] Step F: 2R-[1R (or S)-(Benzyloxy-formyl-amino)-ethyl]-hexanoicacid

[0384] N-[2R-(4S-Benzyl-2-oxo-oxazolidine-3-carbonyl)-1R (orS)-methyl-hexyl]- N-benzyloxy-formamide (1.50 g, 3.31 mmol) wasdissolved in THF (25 ml) and water (5 ml) and the solution was cooled inan ice-water bath. Hydrogen peroxide solution (27% w/w), 13.26 mmol) wasadded followed immediately by lithium hydroxide (167 mg, 3.98 mmol). Thereaction was allowed to warm to room temperature and stirred for afurther 3 hours. The solution was cooled again before addition of sodiumnitrite (0.92 g, 13.3 mmol). After 10 minutes, most of the solvent wasremoved under reduced pressure to leave a white paste which waspartitioned between ethyl acetate (25 ml) and 1 M sodium carbonate (30ml). The organic layer was washed with more sodium carbonate solutionand the combined aqueous extracts were washed with ethyl acetate. Theaqueous layer was cooled and acidified with 1 M hydrochloric acid andextracted twice with ethyl acetate.

[0385] The combined organic layers were washed with brine, dried overanhydrous magnesium sulphate, filtered and evaporated to provide thetitle compound as a green oil (839 mg, 86%). ¹H-NMR; δ (CDCl₃, mixtureof a-diastereoisomers and rotamers), 8.40-7.64 (2H, br m), 7.48-7.27(5H, m), 5.23-4.80 (2H, m), 4.16 (1H, br m), 2.79 (1H, m), 1.67-1.47(2H, m), 1.47-1.18 (7H, m) and 0.95-0.82 (3H, m).

[0386] Step G: 2R-[l R (or S)-(Benzyloxy-formyl-amino)-ethyl]-hexanoicacid (1S-dimethylcarbamoyl-2,2-dimethyl-propyl)-amide

[0387] 2R-[1R (or S)-(Benzyloxy-formyl-amino) ethyl]-hexanoic acid (839mg, 2.86 mmol), tert-leucine N,N-dimethyl amide (498 mg, 3.15 mmol) andEDC (658 mg, 3.43 mmol) were dissolved together in DMF (15 ml) and acatalytic amount of HOAt (60 mg) was added. The solution was left tostir for several days at room temperature. The solvent was removed underreduced pressure and the remaining oil was partitioned between ethylacetate and 1 M hydrochloric acid (75 ml). The organic layer was washedsuccessively with 1 M hydrochloric acid, 1 M sodium carbonate and brine,dried over anhydrous magnesium sulphate filtered and evaporated to leavea yellow foam (1.08 g, 82%). ¹H-NMR; δ (CDCl₃, mixture ofa-diastereoisomers and rotamers), 8.13 (1H, br m), 7.52-7.31 (5H, m),6.28 (1H, br m), 5.36-4.67 (3H, br m), 4.09 (1H, br m), 3.14 (3H, s),2.95 (1 .2H, s), 2.93 (1.8H, s), 2.48 (1H, br m), 1.61-1.04 (9H, m),0.99 (3.6H, s), 0.95 (5.4H, s) and 0.89-0.75 (3H, m).

[0388] Step H: 2R-[1R (or S)-(Formyl-hydroxy-amino)-ethyl]-hexanoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-propyl)-amide

[0389] 2R-[1R (or S)-(Benzyloxy-formyl-amino)-ethyl]-hexanoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-propyl)-amide (200 mg, 0.46 mmol) wasdissolved in methanol (15 ml) and placed under a blanket of argon. Asuspension of 10% palladium on charcoal (20 mg) in ethyl acetate wasadded and the mixture was stirred under an atmosphere of hydrogen for 3hours. The catalyst was removed by filtration and the filtrate wasevaporated to leave a colourless oil (163 mg, quant.). The twodiastereoisomeric products were separated by preparative HPLC.

[0390] Diastereoisomer A (27 mg): ¹H-NMR; δ (CDCl₃, mainly one rotamer),8.67 (0.9H, br s), 8.33 (0.1H, br s), 7.92 (1H, s), 6.74 (0.1H, br m),6.54 (0.9H, d, J=9.4 Hz), 4.93 (0.9H, d, J=9.4 Hz), 4.64 (0.1H, br m),3.89 (1H, qd, J=6.6, 2.6 Hz), 3.16 (3H, s), 2.96 (3H, s), 2.62-2.48 (1H,m), 1.52-1.06 (6H, m), 1.35 (3H, d, J=6.6 Hz), 1.00 (9H, s) and 0.82(3H, t, J=6.9 Hz). ¹³C-NMR; δ (CDCl₃), 173.0,171.3,57.2,54.4, 50.4,38.4,35.6,29.9, 29.1, 26.6, 22.5,17.2 and 13.9. LRMS: +ve ion 366[M+Na], −ve ion 342 [M−H].

[0391] Diastereoisomer B (42 mg): ¹H-NMR; δ (CDCl₃, mixture ofrotamers), 9.15 (0.6H, s), 8.60 (0.4H, br s), 8.42 (0.6H, s), 7.84(0.4H, s), 6.83 (0.6H, d, J=9.2 Hz), 6.55 (0.4H, d, J=9.4 Hz), 4.91(0.6H, d, J=9.2 Hz), 4.89 (0.4H, d, J=9.4 Hz), 4.69 (0.6H, qd, J=7.0,4.3 Hz), 3.92 (0.4H, dq, J=9.1, 6.8 Hz), 3.15 (3H, s), 2.97 (1.8H, s),2.95 (1.2H, s), 2.59 (0.4H, td, J=9.8, 4.3 Hz), 2.39 (0.6H, td, J=7.4,4.3 Hz), 1.92-1.07 (6H, m), 1.37 (1 .2H, d, J=6.8 Hz), 1.31 (1.8H, d,J=7.0 Hz), 1.01 (5.4H, s), 0.96 (3.6H, s), 0.85 (1.8H, t, J=7.2 Hz) and0.83 (1.2H, t, J=7.2 Hz). ¹³C-NMR; δ (CDCl₃, mixture of rotamers),175.7, 173.2,171.3,170.7,56.7, 55.0, 54.4,53.2,50.8,49.9,38.3,35.7,35.6, 35.5,35.4,30.3, 29.5,29.3, 26.5, 26.4,22.5,22.4,16.0,15.4 and 13.8. LRMS: +ve ion 366 [M+Na], -ve ion 342 [M−H].

EXAMPLE 70

[0392]N-cyclohexyl-2-{2-[(formyl-hydroxy-amino)-methyl]-3-phenyl-propionylamino}-3,3-dimethyl-butyramide

[0393] Stock solutions of 1 M ammonia in methanol (1 ml, 1 mmol) and 1 Mtrimethylacetaldehyde in methanol (1 ml, 1 mmol) were mixed in a boilingtube and allowed to stand for 1 hour. A 1 M solution of cyclohexylisocyanide in methanol (1 ml, 1 mmol) was added followed by 0.5 M2RS-[(benzyloxy-formyl-amino)-methyl]-hexanoinc acid in methanol (2 ml,1 mmol). The reaction mixture was allowed to stir at room temperaturefor 2 days. The solvent was removed using a Savant Speedvac and thereaction mixture was crystallised from ethylacetate-hexane to provide2-{2-[(benzyloxy-formyl-amino)-methyl]-3-phenyl-propionylamino}-N-cyclohexyl-3,3-dimethyl-butyramideas a white solid (93 mg, 18%), which was deprotected by catalytictransfer hydrogenolysis (hydrogen gas, 10% palladium on charcoal,methanol-ethyl acetate) to provide the title compound (75 mg, 99%).White solid. LRMS: +ve ion 440 [M+Na], 418 [M+H], −ve ion 416 [M−H].

[0394] The compounds of Examples 71 to 77 were prepared in parallelusing the Ugi 4 component condensation reaction, as described above. Allproducts were obtained in >85% purity as determined by HPLC.

EXAMPLE 71

[0395]2-{2-[(Formyl-hydroxy-amino)-methyl]-3-phenyl-propionylamino}-3,3-dimethyl-hexanoicAcid Cyclohexyl Amide

[0396] White solid (90 mg). ¹H-NMR; δ (CD₃OD), 7.82 (1H, s), 7.29-7.08(5H, m), 4.20 (1H, d, J=5.0 Hz), 3.89 (1H, m), 3.19 (1H, m), 2.95-2.67(2H, m), 1.88-1.58 (5H, br m), 1.44-1.05 (9H, br m) and 0.89 (9H, s).LRMS: +ve ion 468 [M+Na], 446 [M+H], −ve ion 444 [M−H].

EXAMPLE 72

[0397]2-{2-[(Formyl-hydroxy-amino)-methyl]-3-phenyl-propionylamino}-3,3-dimethyl-hexanoicAcid Phenylmethyl Amide

[0398] White solid (77 mg). ¹H-NMR; δ (CD30D), 7.82 (1H, s), 7.35-7.11(1 OH, m), 4.38-4.19 (3H, m), 3.85 (1H, m), 3.52 (1H, m), 2.97-2.63 (3H,m), 1.37-1.11 (4H, m) and 0.93-0.78 (9H, m). LRMS: +ve ion 476 [M+Na],454 [M+H].

EXAMPLE 73

[0399]2-{2-[(Formyl-hydroxy-amino)-methyl]-3-phenyl-propionylamino}-3,3-dimethyl-butyricacid tert-butyl Amide

[0400] White solid (47 mg). ¹H-NMR; δ (CD₃OD), 7.82 (1H, s), 7.45 (1H,m), 7.30-7.09 (5H, m), 4.12(1H, d, J=7.2 Hz), 3.89(1H, m), 3.41 (1H, m),3.15(1H, m), 2.97-2.68 (2H, m), 1.28 (9H, s) and 0.92 (9H, s). LRMS: +veion 414 [M+Na], 392 [M+H], −ve ion 390 [M−H].

EXAMPLE 74

[0401]2-{2-[(formyl-hydroxy-amino)-methyl]-3-phenyl-propionylamino}-3,3-dimethyl-hexanoicacid (1,1,3,3-tetramethyl)-butyramide

[0402] White solid (65 mg). ¹H-NMR; 8 (CD₃OD), 7.79 (1H, s), 7.42-7.21(1H, m), 7.20-7.10 (5H, m), 4.23 (1H, d, J=9.1 Hz), 3.86 (1H, m), 3.51(1H, m), 3.23 (1H, m), 3.00-2.56 (2H, m), 1.50-1.15 (12H, m) and1.02-0.83 (18H, m). LRMS: +ve ion 498 [M+Na], 476 [M+H, −ve ion 474[M−H].

EXAMPLE 75

[0403]N-(Cyclohexyl-cyclohexylcarbamoyl-methyl)-2-[(formyl-hydroxy-amino)-methyl]-3-phenyl-propionamide

[0404] White solid (98 mg). ¹H-NMR; δ (CD₃OD), 7.38-7.08 (5H, m), 4.01(1H, m), 3.81 (1H, m), 3.68-3.35 (2H, m), 3.15 (1H, m), 2.98-2.65 (2H,m), 1.88-1.49 (1 OH, br m) and 1.45-0.83 (1 1H, br m). LRMS: +ve ion 466[M+Na], 444 [M+H], −ve ion 442 [M−H].

EXAMPLE 76

[0405] N-(Cyclohexyl-phenylmethylcarbamoyl-methyl)-2-[(formyl-hydroxy-amino)-methyl]-3-phenyl-propionamide

[0406] White solid (34 mg). ¹H-NMR; δ (CD₃OD),7.35-7.10 (10H, m),4.44-4.23 (2H, m), 4.05 (1H, m), 3.87-3.35 (2H, m), 3.09 (1H, m),2.85-2.72 (2H, m), 1.65-1.46 (4H, m), 1.38-0.93 (5H, br m) and 0.75-0.51(2H, br m). LRMS: +ve ion 474 [M+Na], −ve ion 450 [M−H].

EXAMPLE 77

[0407]N-[Cyclohexyl-(1,1,3,3-tetramethyl-butylcarbamoyl)-methyl]-2-[(Formyl-hydroxy-amino)-methyl]-3-phenyl-propionamide

[0408] White solid (51 mg). ¹H-NMR; δ (CD₃OD), 7.80 (1H, s), 7.36-7.10(5H, m), 4.05 (1H, m), 3.85 (1H, m), 3.49 (1H, m), 3.15 (1H, m), 2.91(1H, m), 2.68 (1H, m), 1.90 (1H, m), 1.80-1.48 (7H, m), 1.40-1.12 (1OH,m) and 1.08-0.83 (1OH, m). LRMS: +ve ion 496 [M+Na], 474 [M+H], −ve ion472 [M−H].

BIOLOGICAL EXAMPLE A

[0409] Demonstration of Antibacterial Effect of Compound 1 (Example 1)and Compound 2 (Example 13).

[0410] a).

[0411] Minimal inhibitory concentrations (MIC) of inhibitors against E.coli strain DH5α (Genotype; F-φ80dlacZΔM15Δ(IacZYA-argF)U169 deoR recAlendAl hsdRl 7(r_(k)-,m_(k) ⁺)phoA supE44λ⁻ thi-1 gyrA96 re/A1) obtainedfrom GibcoBRL Life Technologies, Enterobacter cloacae (American TypeCulture Collection number 13047), Klebsiella pneumoniae (American TypeCulture Collection number 13883) or Staphylococcus capitis (AmericanType Culture Collection number 35661) were determined as follows. Stocksolutions of test compound (Compounds 1 and 2 from Examples 1 and 2respectively (both isomer A)) and three standard laboratory antibiotics,carbenicillin (Sigma, catalogue No. C3416), kanamycin (Sigma, catalogueNo. K4000) and chloramphenicol (Sigma, catalogue No. C1919), wereprepared by dissolution of each compound in dimethylsulfoxide at 10 mM.For the determination of the minimal inhibitory concentration, two foldserial dilutions were prepared in 2xYT broth (typtone 16 g/1, yeastextract 10 g/1, sodium chloride 5 g/1 obtained from BIO 101 Inc, 1070Joshua Way, Vista, Calif. 92083, USA) to yield 0.05 mlcompound-containing medium per well. Inocula were prepared from culturesgrown overnight in 2xYT broth at 37° C. Cell densities were adjusted toabsorbance at 660nm (A₆₆₀) =0.1; the optical density-standardizedpreparations were diluted 1:1000 in 2xYT broth; and each well inoculatedwith 0.05 ml of the diluted bacteria. Microtiter plates were incubatedat 37° C. for 18 hours in a humidified incubator. The MIC (μM) wasrecorded as the lowest drug concentration that inhibited visible growth.TABLE 1 MIC μM com- com- chloramphe- pound pound carbenicillin nicolkanamycin 1 2 E. coli 25 3.12 12.5 12.5 6.25 DH5α Staphylococ <1.56 6.25<1.56 100 25 cus capitis Enterobacter >200 25 50 50 25 cloacaeKlebsiella 200 12.5 25 25 12.5 pneumoniae

[0412] b).

[0413] Minimal inhibitory concentrations (MIC) of inhibitors againstMycobacterium ranae (American Type Culture Collection number 110),Pseudomonas aeruginosa (American Type Culture Collection number 9027),Klebsiella pneumoniae (American Type Culture Collection number 10031),Helicobacter pylori (American Type Culture Collection number 43504),clinical isolates of aminoglycoside and erythromycin resistantStreptococcus pneumoniae and methicillin-resistant (MR) Staphylococcusaureus (American Type Culture Collection number 33591) were determinedas follows. Stock solutions of test compounds 1 and 2 (isomer A foreach) and three standard laboratory antibiotics, gentamycin (G),ampicillin (A) and erythromycin (E), were prepared by dissolution ofeach compound at 10 mg/ml in dimethylsulfoxide. Methods used were as fora) except that the medium of Mycobacterium ranae was used with BrainHeart Infusion broth (GIBCO) and incubated at 37° C. for 48 hours,Staphylococcus aureus (MR), Klebsiella pneumoniae, and Pseudomonasaeruginosa were used with Nutrient Broth (DIFCO) and incubated at 37° C.for 20 hours, Helicobacter pylori was used with Columbia agar base(OXOID) containing 7% sheep blood and incubated at 35° C. for 72 hoursand, Streptococcus pneumoniae was used with tryptic soy broth (DIFCO)containing 7% calf serum and incubated at 37° C. for 48 hours. The MIC(μg/ml) was recorded as the lowest drug concentration that inhibitedvisible growth.

[0414] Positive vehicle control (1% DMSO; no test agent) caused growthof all microorganisms.

[0415] Negative blank control (absence of microorganisms; +test agent)revealed no growth of microorganisms. TABLE 2 MIC μg/ml antibioticcompound 1. compound 2. G A E M. ranae 0.78 0.2 0.2 nd nd S. aureus (MR)3.13 1.56 0.78 nd nd K. pneumoniae 0.2 0.1 0.39 nd nd P. aeruginosa 12.512.5 0.78 100 nd H. pylori 0.1 0.1 0.78 0.1 nd S. pneumoniae 50 12.5 1003.13 100

[0416] In another experiment, minimal inhibitory concentrations ofcompounds 1 and the product of Example 13 (compound 3) against a rangeof Gram-positive and Gram-negative bacteria were determined using theMicrodilution Broth Method according to the approved standard of theNational Committee for Clinical Laboratory Standards procedure (Methidsfor dilution antimicrobial susceptibility tests for bacteria that growaerobically-Fourth Edition ISBN 1-56238-309-4) Activity againstGram-positive bacteria MIC μg/ml Bacterial Strain Compound 3 Compound 1Vancomycin Staphylococcus aureus 8 32 0.25 ATCC 29213 MSSAStaphylococcus aureus 16 16 0.5 ATCC 25923 MSSA Staphylococcus aureus 48 0.5 ATCC 6538 MSSA Staphylococcus 4 8 0.5 epidermidis ATCC 1228Staphylococcus 2 8 0.5 epidermidis ATCC 27626 Enterococcus faecalis 3232 1 ATCC 29212 Enterococcus faecalis 8 128 >128 (Vancomycin resistantstrain) Micrococcus luteus 0.5 0.5 0.25 ATCC 9341

[0417] Activity against Gram-negative bacteria MIC μg/ml BacterialStrain Compound 3 compound 1 Ciprofloxacin Escherichia coli 4 4 <0.125ATCC 25922 Escherichia coli 4 4 <0.125 ATCC 12014 Pseudomonas 128 >128<0.125 aeruginosa ATCC 27853 Enterobacter cloacae 32 32 <0.125 ATCC13047 Morganella morganii >128 128 <0.125 ATCC 36030 Klebsiellapneumoniae 16 16 <0.125 ATCC 13883

[0418] The activities of compound 3 and the product of Example 14(compound 4) against a multi-resistant Enterococcus faecalis clinicalisolate, assessed by the method used for the immediately precedingresults, are set out in the following table, and compared with theresults obtained by the same method for known antibacterial agents:Activity against a multi-resistant Enterococcus faecalis clinicalisolate: MIC μg/ml Bacterial Strain Cpd 3 Cpd 4 Ampicillin CeftazidimeImipenem Erythromycin Ciprofloxacin Vancomycin Enterococcus 32 8 0.5 1281 2 0.5 1 faecalis ATCC 29212 Enterococcus 84 >128 >128 >128 >128 >128 >128 faecalis Vancomycin resistant strain

BIOLOCICAL EXAMPLE B

[0419] i) Cloning of the Escherichia coli PDF Gene

[0420] The E. coli PDF gene was cloned in pET24a(+) (designatedpET24-PDF) and was used to transform BL2l DE3 cells from Novagen Inc,(Madison, Wis.). Clones were selected at 37° C. on YT agar plates (8 g/ltyptone, 5 g/yeast extract, NaCl 5 g/l, agar 15 g/l) supplemented with30 μg/ml kanamycin.

[0421] ii) Expression of PDF

[0422] A 20 ml overnight culture of BL21 DE3 cells harbouring pET24-PDFwas used to infect 500 ml 2× YT broth (16 g/l typtone, log/l yeastextract, NaCl 5 g/l) containing 30 ug/ml kanamycin in a 2 litre baffledflask and grown at 37° C. with shaking to an OD₆₀₀ 0.6. The culture wasthen induced by adjusting the medium to 1.0 mM isopropyl β-Dthiogalactopyranoside (IPTG). The induction was allowed to proceed for afurther 3 hours at 37° C., the cells were harvested by centrifugationand the cell pellet washed with 250 ml phosphate buffered saline (PBS)and the pellet stored at −70° C.

[0423] iii) Preparation of Soluble Protein Fraction.

[0424] The cells from a 1 litre expression were resuspeneded in 2×25 mlof ice cold phosphate buffered saline. The cell suspension was sonicatedon ice using an MSE Soniprep 150 fitted with a medium probe and at anamplitude of 20-25 microns in 6×20 second pluses. The resultingsuspension was then cleared by centrifugation at 20,000× g for 15minutes. The supernatant was then used for further purification of theenzyme.

[0425] iv) PDF Purification

[0426]E. coli lysate from a 11 culture in phosphate buffered saline(PBS) were adjusted to 2M ammonium sulphate. A 15 ml phenyl sepharosecolumn was equilibrated with PBS/2M ammonium sulphate at 4° C. Thelysate was loaded on the column and washed with equilibration buffer.The column was eluted by reducing the ammonium sulphate concentrationfrom 2M to 0M over 10 column volumes. 5 ml fractions were collected andanalysed by SDS-PAGE. The fractions containing the majority of the 20kDa PDF were pooled. The pooled fractions were concentrated using a 3kDa cutoff membrane to a volume of 5 ml. The fraction was then loadedonto a Superdex 75 (size exclusion chromatography) column equilibratedin PBS. The concentrated PDF pool eluted at one ml/min at 4° C. and 5 mlfractions collected and analysed by SDS-PAGE. The purest fractions werepooled and stored at −70° C.

[0427] (v) PDF in Vitro Assay

[0428] The assay was performed in a single 96 well plate in a finalvolume of 100 μl containing:

[0429] 20 μl PDF (4 μg/ml)

[0430] 20 μg 100 mM Hepes pH 7.0+1 M KCl+0.05% Brij

[0431] 10 μl serial dilution of test compound in 20% DMSO

[0432] 50 μl formyl-Met-Ala-Ser (8 mM)

[0433] The assay was incubated at 37° C. for 30 minutes. The free aminogroup of the deformylated (Met-Ala-Ser) product was detected usingfluorescamine, by the following additions:

[0434] 50 μl 0.2M borate pH 9.5

[0435] 50 μl fluorescamine (150 μg/ml in dry dioxane)

[0436] Fluorescence was quantified on SLT Fluostar plate reader using anexcitation wavelength of 390 nM and an emission wavelength of 485 nM.Standard control reactions are a no inhibitor reaction which providesthe zero inhibition figure and a no enzyme and no inhibitor reactionwhich provides the 100% inhibition figure. The data was analysed byconversion of the fluorescence units to % inhibition and the inhibitorconcentration plotted against % inhibition. The data was fitted to asigmoidal function: y=A+((B−A)/(1+((C/x)^(D)))), wherein A representszero inhibition, B represents 100% inhibition and C represents the IC₅₀.D represents the slope. The IC₅₀ represents the concentration ofinhibitor (nM) required to decrease enzyme activity by 50%.

[0437] Compounds of the invention were found to inhibit bacterial PDF invitro. In addition, actinonin (Sigma Cat. No. A-6671) was also found toinhibit bacterial PDF in vitro.

BIOLOGICAL EXAMPLE C

[0438] Demonstration That Compound 2 Inhibits PDF in Vivo.

[0439] 1 Blocking the tRNAi-Met transformylation Reaction ConfersResistance to Compound 2 (Diastereomer/Isomer A).

[0440] Trimethoprim specifically inhibits dihydrofolate reductase,thereby depressing the pools of tetrahydrofolate (THF) derivatives,including formyl tetrahydrofolate (fTHF), the substrate of themethionyl-tRNA formyltransferase (EC_(2.1.2.9)). If all essentialmetabolites whose biosynthesis involves THF derivatives, egpantothenate, methionine, glycine, purine nucleotides and thymidine aresupplied exogenously in the form of precursor compounds in rich mediumsupplemented with thymidine, then bacteria grown in rich medium plusthymidine (0.3 mM) and trimethoprim (100 μg/ml) can synthesize all thechemical components of normal cells except f-Met-tRNA (Baumstark et al.,J. Bacteriol. 129:457-471, 1977). Unformylated Met-tRNAi is usedinstead, resulting in the formation of polypeptides devoid of a formylgroup at their N-terminus, independently of the action of deformylase.As predicted by the inventors, DH5α cells grown in LB medium (typtone 10g/l, yeast extract 5 g/l NaCl log/l pH7.5) supplemented withtrimethoprim and thymidine, were found to be resistant to compound 2(diastereomer A). The demonstration that cells that undergo the normalformylation process on expressed proteins are inhibited by compound 2Awhereas, unformylated proteins, as produced in the cells grown underthese conditions, are not inhibited by compound 2A demonstrates thatcompound 2A is likely to work by inhibiting the deformylation reactioncarried out by PDF. TABLE 3 minimal inhibitory growth conditionsconcentration μM LB 15 LB trimethoprim (100 μg/ml), >200 thymidine (0.3mM)

[0441] 2 Treatment of Bacteria With Compound 2A Leads to theAccumulation of N-Terminally Blocked Proteins.

[0442] If the compounds of the invention do actually inhibit PDF invivo, then a consequence of treatment of bacteria with compound 2(Example 2, diastereomer A) will be the accumulation of N-formylmethionine at the N-terminus of newly synthesised proteins. Suchproteins will be N-terminally blocked and will be unable to be used as asubstrate for N-terminal sequencing by Edman degradation chemistry.

[0443] To test this hypothesis a desired protein is expressed in thepresence or absence of the test compound. The protein is isolated,purified and then subjected to Edman degradation protein sequencingusing techniques known to the person skilled in the art.

[0444] Bacterial cells transformed with an expression vector allowingexpression of the human calpain small regulatory subunit, were grown toan OD₆₀₀ of 0.6 and then subjected to IPTG to induce expression of theheterologous protein in the presence of 200 μM compound 2A, in thepresence of 240 μM carbenicillin or, in the presence of vehicle controlfor 2.5 hours. Protein extracts were separated by SDS-PAGE, the calpainsubunit eluted and the protein sequence determined by Edman degradationchemistry using the ABI automated protein sequencer. Equal quantities ofprotein were sequenced. The inventors found that the yield of thecompound 2A treated protein was significantly reduced by 85% compared tovehicle and carbenicillin treated controls.

[0445] Calpain small regulatory subunit was cloned from messenger RNAobtained from a gastric tumour biopsy using the InVitrogen Micro FastTrack™ mRNA isolation kit version 2.2 (catalogue number Kl520-02). CopyDNA from this mRNA was synthesised using the Promega Riboclone™ cDNAsynthesis system M-MLV-RT(H−), NotI (Promega, Catalogue number C1660)according to the manufactures instructions. Two oligonucleotide primersfor use in the polymerase chain reaction (PCR) were synthesised byApplied Biosystems, Inc., Custom Services, based on the publishedcalpain small subunit sequence (EMBL Accession number X04106).

[0446] The HindIII/XhoI calpain fragment was then cloned into HindIIIand XhoI digested expression vector pET24d(+) from Novagen Inc,(Madison, Wis., USA) using standard procedures. The ligation mixture wasused to transform competent DH5αcells (Life Technologies, Inc, GrandIsland, N.Y., USACat # 18265-017). Colonies were selected by growthovernight at 37° C. on YT plates plus 30 μg/ml kanamycin. Plasmid DNAwas prepared as using the Promega Plus SV miniprep kit and clones withthe calpain insert were identified using standard procedures. The DNAsequence was confirmed using the PE Applied Biosystems cycle sequencingas described above.

[0447] The E. coli gene cloned in pET24d(+) (designated pET24-CANS) wasused to transform BL21 DE3 cells from Novagen Inc, (Madison, Wis.).Clones were selected at 37° C. on YT agar plates (8 g/l typtone, 5g/yeast extract, NaCl 5 g/l, agar 15 g/l) supplemented with 30 μg/mlkanamycin.

We claim:
 1. A compound of formula (I) or a pharmaceutically orveterinarily acceptable salt thereof

R₁ represents hydrogen, C₁-C₆ alkyl, or C₁-C₆ alkyl substituted by oneor more halogen atoms; R₂ represents a group R₁₀—(ALK)_(m)— wherein R₁₀represents hydrogen, or a C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, acycloalkyl, aryl, or heterocyclyl group, any of which may beunsubstituted or substituted by (C₁-C₆)alkyl, (C₁-C₆)alkoxy, hydroxy,mercapto, (C₁-C₆)alkylthio, amino, halo, trifluoromethyl, nitro, —COOH,—CONH₂, —COOR^(A), —NHCOR^(A), —CONH^(A), —NH^(A), —NR^(A)R^(B), or—CONR^(A)R^(B) wherein R^(A) and R^(B) are independently a (C₁-C₆)alkylgroup, ALK represents a straight or branched divalent C₁-C₆ alkylene,C₂-C₆ alkenylene, C₂-C₆ alkynylene radical, and may be interrupted byone or more non-adjacent —NH—, —O—or —S—linkages, and m represents 0 or1; A represents (i) a group of formula (IA), (IB), (IC) or (ID)

wherein: R₃ represents hydrogen and R₄ represents the side chain of anatural or non-natural alpha amino acid or R₃ and R₄ when taken togetherwith the nitrogen and carbon atoms to which they are respectivelyattached form an optionally substituted saturated heterocyclic ring of 5to 8 atoms which ring is optionally fused to a carbocyclic or secondheterocyclic ring, R₅ and R₆, independently represent hydrogen, oroptionally substituted C₁-C₈ alkyl, cycloalkyl, aryl(C₁-C₆ alkyl),non-aromatic heterocyclic, or heterocyclic(C₁-C₆ alkyl), or R₅ and R₆when taken together with the nitrogen atom to which they are attachedform an optionally substituted saturated heterocyclic ring of 3 to 8atoms which ring is optionally fused to a carbocyclic or secondheterocyclic ring, and R₇ represents hydrogen, C₁-C₆ alkyl, or an acylgroup. PROVIDED THAT (i) when A is a group of formula (IA) or (IB) andR₂ is C₂-C₅ alkyl then R₄ is not the side chain of a natural alpha aminoacid or the side chain of a natural alpha-amino acid in which anyfunctional substituents are protected, any amino groups are acylated,and any carboxyl groups are esterified; (ii) when A is a group offormula (IA) or (1B) then R₄ is not a bicyclicarylmethyl group; and(iii) when A is a group of formula (IA) and R₂ is cyclopropylmethyl,cyclobutylmethyl or cyclopentylmethyl and one of R₅ and R₆ is hydrogen,then R₄ is not tert-butyl.
 2. A compound as claimed in claim 1 wherein:R₁ represents hydrogen, or C₁-C₆ alkyl; R₂ represents a group R₁₀—(ALK)—wherein R₁₀ represents hydrogen, a C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, a cycloalkyl, aryl, or heteroaryl group, any of which may beunsubstituted or substituted by (C₁-C₆)alkyl, (C₁-C₆)alkoxy, hydroxy,mercapto, (C₁-C₆)alkylthio, amino, halo, trifluoromethyl, nitro, —COOH,—CONH₂, —COOR^(A), —NHCOR^(A),—CONH^(A), —NH^(A), —NR^(A)R^(B), or—CONR^(A)R^(B) wherein R^(A) and R^(B) are independently a (C₁-C₆)alkylgroup, and ALK represents a straight or branched divalent C₁-C₆alkylene, C₂-C₆ alkenylene, C₂-C₆ alkynylene radical, and may beinterrupted by one or more non-adjacent —NH—, —O— or —S—linkages; R₃represents hydrogen or C₁-C₆ alkyl, R₄ represents the side chain of anatural or non-natural alpha amino acid, R₅ and R₆, independentlyrepresent hydrogen or C₁-C₆ alkyl, or R₅ and R₆ when taken together withthe nitrogen atom to which they are attached form an optionallysubstituted saturated heterocyclic ring of 3 to 8 atoms, and R₇represents hydrogen or an acyl group; PROVIDED THAT when A is a group offormula (IA) then (i) R₄ is not the side chain of a natural alpha aminoacid or the side chain of a natural alpha-amino acid in which anyfunctional substituents are protected, any amino groups are acylated,and any carboxyl groups are esterified, (ii) R₄ is not a fusedbicyclicarylmethylene group, and (iii) R₄ is not tert-butyl when R₂ iscycloalkyl(C₁-C₆ alkyl).
 3. The compound as claimed in claim 1 whereinin the compound of formula (I) R₁ is hydrogen.
 4. The compound asclaimed in claim 1 wherein in the compound of formula (I) R₂ is:optionally substituted C₁-C₈ alkyl, C₃-C₆ alkenyl, C₃-C₆ alkynyl orcycloalkyl; phenyl(C₁-C₆ alkyl)-, phenyl(C₃-C₆ alkenyl)- or phenyl(C₃-C₆alkynyl)- optionally substituted in the phenyl ring; cycloalkyl(C₁-C₆alkyl)-, cycloalkyl(C₃-C₆ alkenyl)- or cycloalkyl(C₃-C₆alkynyl)-optionally substituted in the cycloalkyl ring;heterocyclyl(C₁-C₆ alkyl)-, heterocyclyl(C₃-C₆ alkenyl)- orheterocyclyl(C₃-C₆ alkynyl)- optionally substituted in the heterocyclylring; or CH₃(CH₂)_(p)O(CH₂)_(q)— or CH₃(CH₂)_(p)S(CH₂)_(q)—, wherein pis 0, 1, 2 or 3 and q is 1, 2 or
 3. 5. The compound as claimed in claim1 wherein in the compound of formula (I) R₂ is methyl, ethyl, n- oriso-propyl, n- or iso-butyl, n-pentyl, iso-pentyl 3-methyl-but-1-yl,n-hexyl, n-heptyl, n-acetyl, n-octyl, methylsulfanylethyl,ethylsulfanylmethyl, 2-methoxyethyl, 2-ethoxyethyl, 2-ethoxymethyl,3-hydroxypropyl, allyl, 3-phenylprop-3-en-1-yl, prop-2-yn-1-yl,3-phenylprop-2-yn-1-yl, 3-(2-chlorophenyl)prop-2-yn-1-yl, but-2-yn-1-yl,cyclopentyl, cyclohexyl, cyclopentylmethyl, cyclopentylethyl,cyclopentylpropyl, cyclohexylmethyl, cyclohexylethyl, cyclohexylpropyl,furan-2-ylmethyl, furan-3-methyl, tetrahydrofuran-2-ylmethyl,tetrahydrofuran-2-ylmethyl, piperidinylmethyl, phenylpropyl,4-chlorophenylpropyl, 4-methylphenylpropyl, 4-methoxyphenylpropyl,benzyl, 4-chlorobenzyl, 4-methylbenzyl, or 4-methoxybenzyl.
 6. Thecompound as claimed in claim 1 wherein in the compound of formula (I) R₂is n-butyl, benzyl or cyclopentylmethyl.
 7. The compound as claimed inclaim 1 wherein in the compound of formula (I) R₃ is hydrogen.
 8. Thecompound as claimed in claim 1 wherein in the compound of formula (I) R₄is: the side chain of a natural a amino acid; or a group —(Alk)_(n)R₉where Alk is a (C₁-C₆)alkylene or (C₂-C₆)alkenylene group optionallyinterrupted by one or more —O—, or —S—atoms or —N(R₁₂)— groups [whereR₁₂ is a hydrogen atom or a (C₁-C₆)alkyl group], n is 0 or 1, and R₉ ishydrogen or an optionally substituted phenyl, aryl, heterocyclyl,cycloalkyl or cycloalkenyl group or (only when n is 1) R₉ mayadditionally be hydroxy, mercapto, (C₁-C₆)alkylthio, amino, halo,trifluoromethyl, nitro, —COOH, —CONH₂, —COOR^(A), —NHCOR^(A), —CONH^(A),—NH^(A), —NR^(A)R^(B), or —CONR^(A)R^(B) wherein R^(A) and R^(B) areindependently a (C₁-C₆)alkyl group; or a benzyl group substituted in thephenyl ring by a group of formula —OCH₂COR₈ where R₈ is hydroxyl, amino,(C₁-C₆)alkoxy, phenyl(C₁-C₆)alkoxy, (C₁-C₆)alkylamino,di((C₁-C₆)alkyl)amino, or phenyl(C₁-C₆)alkylamino; or aheterocyclic(C₁-C₆)alkyl group, either being unsubstituted or mono- ordi-substituted in the heterocyclic ring with halo, nitro, carboxy,(C₁-C₆)alkoxy, cyano, (C₁-C₆)alkanoyl, trifluoromethyl (C₁-C₆)alkyl,hydroxy, formyl, amino, (C₁-C₆)alkylamino, di-(C₁-C₆)alkylamino,mercapto, (C₁-C₆)alkylthio, hydroxy(C₁-C₆)alkyl, mercapto(C₁-C₆)alkyl or(C₁-C₆)alkylphenylmethyl; or a group —CR_(a)R_(b)R_(c) in which: each ofR_(a), R_(b) and R_(c) is independently hydrogen, (C₁-C₆)alkyl,(C₂-C₆)alkenyl, (C₂-C₆)alkynyl, phenyl(C₁-C₆)alkyl, or(C₃-C₈)cycloalkyl; or R_(c) is hydrogen and R_(a) and R_(b) areindependently phenyl or heteroaryl; or R_(c) is hydrogen, (C₁-C₆)alkyl,(C₂-C₆)alkenyl, (C₂-C₆)alkynyl, phenyl(C₁-C₆)alkyl, or(C₃-C₈)cycloalkyl, and R_(a) and R_(b) together with the carbon atom towhich they are attached form a 3 to 8 membered cycloalkyl or a 5- to6-membered heterocyclic ring; or R_(a), R_(b) and R_(c) together withthe carbon atom to which they are attached form a tricyclic ring; orR_(a) and R_(b) are each independently (C₁-C₆)alkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, phenyl(C₁-C₆)alkyl, or a group as defined for R_(c)below other than hydrogen, or R_(a) and R_(b) together with the carbonatom to which they are attached form a cycloalkyl or heterocyclic ring,and R_(c) is hydrogen, —OH, —SH, halogen, —CN, —CO₂H,(C₁-C₄)perfluoroalkyl, —CH₂OH, —CO₂(C₁-C₆)alkyl, —O(C₁-C₆)alkyl,—O(C₂-C₆)alkenyl, —S(C₁-C₆)alkyl, —SO(C₁-C₆)aIkyl, —SO_(2(C) ₁-C₆)alkyl, —S(C₂-C₆)alkenyl, —SO(C₂-C₆)alkenyl, —SO₂(C₂-C₆)alkenyl or agroup —Q—W wherein Q represents a bond or —O—, —S—, —SO— or —SO₂— and Wrepresents a phenyl, phenylalkyl, (C₃-C₈)cycloalkyl,(C₃-C₈)cycloalkylalkyl, (C₄-C₈)cycloalkenyl, (C₄-C₈)cycloalkenylalkyl,heteroaryl or heteroarylalkyl group, which group W may optionally besubstituted by one or more substituents independently selected from,hydroxyl, halogen, —CN, —CO₂H, —CO_(2(C) ₁-C₆)alkyl, —CONH₂,—CONH(C₁-C₆)alkyl, —CONH(C₁-C₆alkyl)₂, —CHO, —CH₂OH,(C₁-C₄)perfluoroalkyl, —O(C₁-C₆)alkyl, —S(C₁-C₆)alkyl, —SO(C₁-C₆)alkyl,—SO₂(C₁-C₆)alkyl, —NO₂, —NH₂, —NH(C₁-C₆)alkyl, —N((C₁-C₆)alkyl)₂,—NHCO(C₁-C₆)alkyl, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,(C₃-C₈)cycloalkyl, (C₄-C₈)cycloalkenyl, phenyl or benzyl.
 9. Thecompound as claimed in claim 1 wherein in the compound of formula (I) R₄is methyl, ethyl, benzyl, 4-chlorobenzyl, 4-hydroxybenzyl, phenyl,cyclohexyl, cyclohexylmethyl, pyridin-3-ylmethyl, tert-butoxymethyl,naphthylmethyl, iso-butyl, sec-butyl, tert-butyl,1-benzylthio-1-methylethyl, 1-methylthio-1-methylethyl,1-mercapto-1-methylethyl, 1-methoxy-1-methylethyl,1-hydroxy-1-methylethyl, 1-fluoro-1-methylethyl, 2-hydroxethyl,2-carboxyethyl, 2-methylcarbamoylethyl, 2-carbamoylethyl, or4-aminobutyl.
 10. The compound as claimed in claim 1 wherein in thecompound of formula (I) R₄ is tert-butyl, iso-butyl, benzyl or methyl.11. The compound as claimed in claim 1 wherein in the compound offormula (I) R₃ and R₄ when taken together with the nitrogen and carbonatoms to which they are respectively attached form an optionallysubstituted saturated heterocyclic ring of 5 to 8 atoms.
 12. Thecompound as claimed in claim 1 wherein in the compound of formula (I) R₃and R₄ form a bridge between the nitrogen and carbon atoms to which theyare attached, said bridge being represented by the divalent radical—(CH₂)₃₋₆—, or —(CH₂)_(r)—O—(CH₂)_(s)—, or —(CH₂)_(r)—S—(CH₂)_(s)—,wherein r and s are each independently 1, 2 or 3 with the proviso thatr+s =2, 3, 4, or
 5. 13. The compound as claimed in claim 1 wherein inthe compound of formula (I) R₅ and R₆ are independently hydrogen,methyl, ethyl, tert-butyl, cyclopentyl, cyclohexyl,1,1,3,3-tetramethylbutyl,benzyl, or 2-hydroxyethyl.
 14. The compound asclaimed in claim 1 wherein in the compound of formula (I) R₅ and R₆ whentaken together with the nitrogen atom to which they are attached form asaturated 5- to 8-membered monocyclic N-heterocyclic ring which isattached via the N atom and which optionally contains -N(R₁₁)- whereinR₁₁ is hydrogen or C₁-C₆ alkyl, benzyl, acyl, or an amino protectinggroup, O, S, SO or SO₂ as a ring member, and/or is optionallysubstituted on one or more C atoms by hydroxy, C₁-C₆ alkyl,hydroxy(C₁-C₆ alkyl)-, C₁-C₆ alkoxy, oxo, ketalised oxo, amino,mono(C₁-C₆ alkyl)amino, di(C₁-C₆ alkyl)amino, carboxy, C₁-C₆alkoxycarbonyl, hydroxymethyl, C₁-C₆ alkoxymethyl, carbamoyl, mono(C₁-C₆alkyl)carbamoyl, di(C₁-C₆ alkyl)carbamoyl, or hydroxyimino.
 15. Thecompound as claimed in claim 1 wherein in the compound of formula (I) R₅and R₆ when taken together with the nitrogen atom to which they areattached form a substituted or unsubstituted 1-pyrrolidinyl,piperidin-1-yl, 1-piperazinyl, hexahydro-1-pyridazinyl, morpholin-4-yl,tetrahydro-1,4-thiazin-4-yl, tetrahydro-1,4-thiazin-4-yl 1-oxide,tetrahydro-1,4-thiazin-4-yl 1,1-dioxide, thiazolidin-3-yl,hexahydroazipino, or octahydroazocino ring.
 16. The compound as claimedin claim 1 wherein in the compound of formula (I) R₅ and R₆ when takentogether with the nitrogen atom to which they are attached form asubstituted or unsubstituted 1-pyrrolidinyl, piperidin-1-yl,1-piperazinyl, hexahydro-1-pyridazinyl, morpholin-4-yl,tetrahydro-1,4-thiazin-4-yl, tetrahydro-1,4-thiazin-4-yl 1-oxide,tetrahydro-1,4-thiazin-4-yl 1,1-dioxide, hexahydroazipino, oroctahydroazocino. Substituted examples of the foregoing are2-(methylcarbamoyl)-1-pyrrolidinyl, 2-(hydroxymethyl)-1-pyrrolidinyl,4-hydroxypiperidino, 2-(methylcarbamoyl)piperidino,4-hydroxyiminopiperidino, 4-methoxypiperidino, 4-methylpiperidin-1 yl,4-benzylpiperidin-1-yl, 4-acetylpiperidin-1-yl, 4-methyl-1-piperazinyl,4-phenyl-1-piperazinyl, 1,4-dioxa-8-azaspiro(4,5)decan-8-yl,hexahydro-3-(methylcarbamoyl)-2-pyridazinyl, andhexahydro-1-(benzyloxycarbonyl)-2-pyridazinyl,decahydroisoquinolin-2-yl, or 1,2,3,4-tetrahydroisoquinolin-2-yl ring.17. The compound as claimed in claim 1 wherein in the compound offormula (I) R₇ is hydrogen, or a group R₂₀C(O)— where R₂₀ is a(C₁-C₆)alkyl group.
 18. The compound as claimed in claim 1 wherein inthe compound of formula (I) R₂₀ is methyl or ethyl.
 19. The compound asclaimed in claim 1 wherein in the compound of formula (I) A represents agroup of formula (IA), R₁ represents hydrogen, R₂ represents n-butyl,benzyl or cyclopentylmethyl, R₃ is hydrogen, R₄ is tert-butyl,iso-butyl, benzyl or methyl, R₅ is hydrogen or methyl and R₆ representsmethyl.
 20. The compound as claimed in claim 1 wherein in the compoundof formula (I) A represents a group of formula (IB), R₁ representshydrogen, R₂ represents n-butyl, benzyl or cyclopentylmethyl, R₃ ishydrogen, and R₄ is tert-butyl, iso-butyl, benzyl or methyl.
 21. 2R or2S-((Formyl-hydroxy-amino)-methyl)-hexanoic acid(1S-dimethylcarbamoyl-ethyl)-amide or a pharmaceutically or veterinarilyacceptable salt thereof.
 22. 2R or2S-((Formyl-hydroxy-amino)-methyl)-3-cyclopentyl-propionic acid(1S-dimethyl-carbamoyl-2,2-dimethyl-propyl)-amide or a pharmaceuticallyor veterinarily acceptable salt thereof.
 23. An antibacterialpharmaceutical or veterinary composition comprising a compound asclaimed claim 1 together with a pharmaceutically or veterinarilyacceptable excipient or carrier.
 24. An antibacterial pharmaceutical orveterinary composition comprising a compound of formula (I) or apharmaceutically or veterinarily acceptable salt thereof

R₁ represents hydrogen, C₁-C₆ alkyl, or C₁-C₆ alkyl substituted by oneor more halogen atoms; R₂ represents a group R₁₀—(X)_(n)—(ALK)_(m)—wherein R₁₀ represents hydrogen, or a C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, a cycloalkyl, aryl, or heterocyclyl group, any of which may beunsubstituted or substituted by (C₁-C₆)alkyl, (C₁-C₆)alkoxy, hydroxy,mercapto, (C₁-C₆)alkylthio, amino, halo, trifluoromethyl, nitro, —COOH,—CONH₂, —COOR^(A), —NHCOR^(A), —CONH^(A), —NH^(A), —NR^(A)R^(B), or—CONR^(A)R^(B) wherein R^(A) and R^(B) are independently a (C₁-C₆)alkylgroup, ALK represents a straight or branched divalent C₁-C₆ alkylene,C₂-C₆ alkenylene, C₂-C₆ alkynylene radical, and may be interrupted byone or more non-adjacent —NH—, —O— or —S—linkages, X represents —NH—,—O— or —S—, and m and n are independently 0 or 1; A represents (i) agroup of formula (IA), (IB), (IC) or (ID)

wherein: R₃ represents hydrogen and R₄ represents the side chain of anatural or non-natural alpha amino acid or R₃ and R₄ when taken togetherwith the nitrogen and carbon atoms to which they are respectivelyattached form an optionally substituted saturated heterocyclic ring of 5to 8 atoms which ring is optionally fused to a carbocyclic or secondheterocyclic ring, R₅ and R₆, independently represent hydrogen, oroptionally substituted C₁-C₈ alkyl, cycloalkyl, aryl(C₁-C₆ alkyl),non-aromatic heterocyclic, or heterocyclic(C₁-C₆ alkyl), or R₅ and R₆when taken together with the nitrogen atom to which they are attachedform an optionally substituted saturated heterocyclic ring of 3 to 8atoms which ring is optionally fused to a carbocyclic or secondheterocyclic ring, and R₇ represents hydrogen, C₁-C₆ alkyl, or an acylgroup. PROVIDED THAT (i) when A is a group of formula (IA) or (IB) andR₂ is C₂-C₅ alkyl then R₄ is not the side chain of a natural alpha aminoacid or the side chain of a natural alpha-amino acid in which anyfunctional substituents are protected, any amino groups are acylated,and any carboxyl groups are esterified; (ii) when A is a group offormula (IA) or (IB) then R₄ is not a bicyclicarylmethyl group; and(iii) when A is a group of formula (IA) and R₂ is cyclopropylmethyl,cyclobutylmethyl or cyclopentylmethyl and one of R₅ and R₆ is hydrogen,then R₄ is not tert-butyl.
 25. A method for the identification ofantibacterial compounds, comprising (i) screening test compounds foractivity as inhibitors of PDF in vitro, (ii) screening those compoundswhich exhibit said PDF inhibitory activity for ability to inhibitbacterial growth.
 26. A method of treating bacterial infection orcontamination by administering to a patient suffering such infection orcontamination, or applying to the site of such infection orcontamination, an antibacterially effective amount of a compound whichinhibits the activity of bacterial PDF enzyme, provided that (i) thecompound is not of formula (Xl) RCO—CH(W)—NH—CO—CH(Y)—CH₂—CO—NH—OH  (XI) wherein, (a) R is a cyclic amino group, Wis hydrogen, methyl, isopropyl, isobutyl or benzyl, and Y is hydrogen,C₁-C₆ alkyl, phenyl, benzyl, 4-chlorophenylmethyl, 4-nitrophenylmethyl,or 4-aminophenylmethyl; or, (b) R is 2-pyridylamino or 2-thiazolylamino,W is isopropyl and Y is n-pentyl; or, (c) R is diethylamino, W is methylor isopropyl and Y is n-pentyl; or (ii) the compound is not onecontaining a divalent piperazin-1,6-diyl group.